Background: Zika virus infection is a threat to at-risk populations, causing major birth defects and serious neurological complications. Development of a safe and efficacious Zika virus vaccine is, therefore, a global health priority. Assessment of heterologous flavivirus vaccination is important given co-circulation of Japanese encephalitis virus and yellow fever virus with Zika virus. We investigated the effect of priming flavivirus naive participants with a licensed flavivirus vaccine on the safety and immunogenicity of a purified inactivated Zika vaccine (ZPIV). Methods: This phase 1, placebo-controlled, double-blind trial was done at the Walter Reed Army Institute of Research Clinical Trials Center in Silver Spring, MD, USA. Eligible participants were healthy adults aged 18–49 years, with no detectable evidence of previous flavivirus exposure (by infection or vaccination), as measured by a microneutralisation assay. Individuals with serological evidence of HIV, hepatitis B, or hepatitis C infection were excluded, as were pregnant or breastfeeding women. Participants were recruited sequentially into one of three groups (1:1:1) to receive no primer, two doses of intramuscular Japanese encephalitis virus vaccine (IXIARO), or a single dose of subcutaneous yellow fever virus vaccine (YF-VAX). Within each group, participants were randomly assigned (4:1) to receive intramuscular ZPIV or placebo. Priming vaccinations were given 72–96 days before ZPIV. ZPIV was administered either two or three times, at days 0, 28, and 196–234. The primary outcome was occurrence of solicited systemic and local adverse events along with serious adverse events and adverse events of special interest. These data were analysed in all participants receiving at least one dose of ZPIV or placebo. Secondary outcomes included measurement of neutralizing antibody responses following ZPIV vaccination in all volunteers with available post-vaccination data. This trial is registered at ClinicalTrials.gov, NCT02963909. Findings: Between Nov 7, 2016, and Oct 30, 2018, 134 participants were assessed for eligibility. 21 did not meet inclusion criteria, 29 met exclusion criteria, and ten declined to participate. 75 participants were recruited and randomly assigned. 35 (47%) of 75 participants were male and 40 (53%) were female. 25 (33%) of 75 participants identified as Black or African American and 42 (56%) identified as White. These proportions and other baseline characteristics were similar between groups. There were no statistically significant differences in age, gender, race, or BMI between those who did and did not opt into the third dose. All participants received the planned priming IXIARO and YF-VAX vaccinations, but one participant who received YF-VAX dropped out before receipt of the first dose of ZPIV. 50 participants received a third dose of ZPIV or placebo, including 14 flavivirus-naive people, 17 people primed with Japanese encephalitis virus vaccine, and 19 participants primed with yellow fever vaccine. Vaccinations were well tolerated across groups. Pain at the injection site was the only adverse event reported more frequently in participants who received ZPIV than in those who received placebo (39 [65%] of 60 participants, 95% CI 51·6–76·9 who received ZPIV vs three [21·4%] of 14 who received placebo; 4·7–50·8; p=0·006). No patients had an adverse event of special interest or serious adverse event related to study treatment. At day 57, the flavivirus-naive volunteers had an 88% (63·6–98·5, 15 of 17) seroconversion rate (neutralising antibody titre ≥1:10) and geometric mean neutralising antibody titre (GMT) against Zika virus of 100·8 (39·7–255·7). In the Japanese encephalitis vaccine-primed group, the day 57 seroconversion rate was 31·6% (95% CI 12·6–56·6, six of 19) and GMT was 11·8 (6·1–22·8). Participants primed with YF-VAX had a seroconversion rate of 25% (95% CI 8·7–49·1, five of 20) and GMT of 6·6 (5·2–8·4). Humoral immune responses rose substantially following a third dose of ZPIV, with seroconversion rates of 100% (69·2–100; ten of ten), 92·9% (66·1–99·8; 13 of 14), and 60% (32·2–83·7, nine of 15) and GMTs of 511·5 (177·6–1473·6), 174·2 (51·6–587·6), and 79 (19·0–326·8) in the flavivirus naive, Japanese encephalitis vaccine-primed, and yellow fever vaccine-primed groups, respectively. Interpretation: We found ZPIV to be well tolerated in flavivirus naive and primed adults but that immunogenicity varied significantly according to antecedent flavivirus vaccination status. Immune bias towards the flavivirus antigen of initial exposure and the timing of vaccination may have impacted responses. A third ZPIV dose overcame much, but not all, of the discrepancy in immunogenicity. The results of this phase 1 clinical trial have implications for further evaluation of ZPIV's immunisation schedule and use of concomitant vaccinations. Funding: Department of Defense, Defense Health Agency; National Institute of Allergy and Infectious Diseases; and Division of Microbiology and Infectious Disease.