The colonization factors (CF) of enterotoxigenic Escherichia coli (ETEC) are being targeted for inclusion in a multi-subunit ETEC vaccine. This study was designed to examine the preclinical safety and immunogenicity of CF CS6, encapsulated in a biodegradable poly(dl-lactide-co-glycolide) (meCS6), and administered in the presence or absence of a mutated heat-labile enterotoxin, LT(R192G), in the non-human primate, Aotus nancymae. A. nancymae were inoculated intranasally (IN) with meCS6 (200 μg; positive control), or intragastrically (IG) with meCS6 (200 or 1000 μg) with or without 2 μg LT(R192G) in three doses given at 2-week intervals. In a second experiment, A. nancymae were inoculated IG with 950 μg of meCS6 with or without 2 μg LT(R192G) in four doses given every 48 h. Blood was collected to assess anti-CS6 and -LT serum immunoglobulin G (IgG) and IgA responses and safety variables (complete blood count and chemistry). Safety parameters were unchanged from baseline following all vaccinations. In Experiment 1, a dose-related serologic response to CS6 was observed; 78.6 and 57.1% of monkeys given 1000 μg meCS6 (n = 14) had a serum IgG and IgA response, respectively, compared to only 28.6% of monkeys given 200 μg meCS6 (n = 14) with a serum IgG and IgA response. No significant effect on the number of responders or the magnitude of responses was observed with the addition of LT(R192G). The three-dose, 2-week regimen with 1000 μg meCS6 was more effective at eliciting an immune response than the four-dose, 48-h regimen with 950 μg meCS6. Results from this study indicate that A. nancymae provide a useful ETEC preclinical safety and immunogenicity model.