Methods: In an observer blind, phase 2 trial, 55 adults were randomized to receive one dose of Ad35. CS.01 vaccine followed by two doses of RTS,S/AS01 (ARR-group) or three doses of RTS, S/AS01 (RRR-group) at months 0, 1, 2 followed by controlled human malaria infection. Results: ARR and RRR vaccine regimens were well tolerated. Efficacy of ARR and RRR groups after controlled human malaria infection was 44% (95% confidence interval 21%-60%) and 52% (25%-70%), respectively. The RRR-group had greater anti-CS specific IgG titers than did the ARR-group. There were higher numbers of CS-specific CD4 T-cells expressing > 2 cytokine/activation markers and more ex vivo IFN-γ enzyme-linked immunospots in the ARR-group than the RRR-group. Protected subjects had higher CS-specific IgG titers than non-protected subjects (geometric mean titer, 120.8 vs 51.8 EU/ml, respectively; P = .001). Conclusions: An increase in vaccine efficacy of ARR-group over RRR-group was not achieved. Future strategies to improve upon RTS,S-induced protection may need to utilize alternative highly immunogenic prime-boost regimens and/or additional target antigens. Trial Registration: ClinicalTrials.gov NCT01366534.