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α-Defensins have been shown to inhibit HIV-1 replication in vitro and may contribute to the overall control of viral replication in vivo . In the present work, we quantitatively measured the levels of α-defensins in the plasma of healthy and experimentally SHIV-infected Macaca fascicularis (cynomolgus monkeys), an animal model of AIDS pathogenesis and vaccine development. Characterization of physiological plasma α-defensins levels was performed in 12 healthy monkeys following longitudinal analysis using an α-defensins ELISA kit currently validated for macaque use. Subsequently, α-defensins levels were quantitatively measured in 23 cynomolgus monkeys during titration protocols following both the mucosal and systemic routes of infection with the pathogenic SHIV89.6P_cy11. A significant increase in plasma α-defensins levels was consistently observed at early time points in all infected animals, regardless of the infection route. Moreover, a positive correlation was observed between viral replication and levels of α-defensins during the acute phase of infection. Interestingly, in the animals infected through the mucosal route, α-defensins levels remained significantly higher at later time points, up to 19 weeks from the infection, while in cynomolgus infected intravenously, α-defensins levels returned to baseline levels by 4 weeks from infection, suggesting that the different route of infection may differently activate the innate immune response.