Poison frogs contain an alkaloid-based chemical defense that is sequestered directly from a diet of alkaloid-containing arthropods. Geographic and temporal variation in alkaloid defense is common in poison frogs and is generally attributed to differences in the availability of alkaloid-containing arthropods. Variable chemical defense in poison frogs may have important consequences for predator-prey interactions, requiring a full understanding of the factors involved in explaining such variation. In the present study, we examine alkaloid variation in the dendrobatid poison frog Oophaga pumilio between males and females on Cayo Nancy (Isla Solarte), located in the Bocas del Toro archipelago of Panama. On average, females contained a significantly larger number and quantity of alkaloids when compared to males. Alkaloid composition varied significantly between males and females, illustrating that chemical defense in this population of O. pumilio is sex-dependent. The variation in alkaloids between sexes is attributed to differences in feeding and behavior between males and females. The majority of alkaloids present in the skin of O. pumilio appear to be of oribatid mite origin, supporting the importance of these dietary arthropods in the chemical defense of poison frogs.