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To achieve global elimination of human rabies from dogs by 2030, evidence-based strategies for effective dog vaccination are needed. Current guidelines recommend inclusion of dogs younger than 3 months in mass rabies vaccination campaigns, although available vaccines are only recommended for use by manufacturers in older dogs, ostensibly due to concerns over interference of maternally-acquired immunity with immune response to the vaccine. Adverse effects of vaccination in this age group of dogs have also not been adequately assessed under field conditions. In a single-site, owner-blinded, randomized, placebo-controlled trial in puppies born to mothers vaccinated within the previous 18 months in a high-mortality population of owned, free-roaming dogs in South Africa, we assessed immunogenicity and effect on survival to all causes of mortality of a single dose of rabies vaccine administered at 6 weeks of age. We found that puppies did not have appreciable levels of maternally-derived antibodies at 6 weeks of age (geometric mean titer 0.065 IU/mL, 95% CI 0.061–0.069; n = 346), and that 88% (95% CI 80.7–93.3) of puppies vaccinated at 6 weeks had titers ≥0.5 IU/mL 21 days later (n = 117). Although the average effect of vaccination on survival was not statistically significant (hazard ratio [HR] 1.35, 95% CI 0.83–2.18), this effect was modified by sex (p = 0.02), with the HR in females 3.09 (95% CI 1.24–7.69) and the HR in males 0.79 (95% CI 0.41–1.53). We speculate that this effect is related to the observed survival advantage that females had over males in the unvaccinated group (HR 0.27; 95% CI 0.11–0.70), with vaccination …