Buckwheat trypsin inhibitor (BTI) is a low molecular weight polypeptide extracted from buckwheat. This study examined the effects of BTI on the lifespan of Caenorhabditis elegans (C. elegans) and investigated the mechanism involved. Our results showed that recombinant BTI (rBTI) extended life expectancy by mimicking calorie restriction (CR) in C. elegans. rBTI promoted formation of reactive oxygen species (ROS) via increasing respiration, induced activities of ROS defense enzymes by activating DAF-16, and increased oxidative stress resistance and survival rates. The inhibition of ROS signal by antioxidants reduced rBTI-mediated longevity by up to 65%. Moreover, it was shown that the disruption of daf-2 abolished the extension of the lifespan and the increased ROS. Taken together, these data indicate that rBTI-mediated longevity mimics CR by down-regulating insulin/IGF-1 signaling (IIS) pathway, implying that BTI has the potential to be a novel anti-aging drug.