Resource availability, predation, and sexual selection have all been shown to play an important role in the ecology and evolution of guppies, Poecilia reticulata, but the role of parasitism has received comparatively little attention. In the present study, we examined natural infection levels of wild-caught guppies from the Upper and Lower Aripo River in Trinidad (UA and LA, respectively) and experimentally infected a subsample of fish with an isogenic line of the ectoparasite Gyrodactylus turnbulli. LA fish showed a superior resistance compared to UA guppies during the primary, but not secondary infection 53 days later. Resistance of LA individuals was consistent across experiments, suggesting immunocompetence has a heritable genetic basis. The efficiency of the immune response of UA fish was not correlated across infections. During primary infections, UA fish were highly susceptible, but their resistance was significantly improved during a secondary infection, highlighting the importance of acquired resistance. We discuss the role of innate and acquired resistance, and place our findings in the context of natural gyrodactylid infections.