Comparative studies documenting a relationship between male gonadal investment and the degree of sperm competition (SC) have usually considered the association between these traits to be driven by qualitative differences in the mating system, such as whether spawning occurs in pairs or groups. However, ecological and demographic differences between conspecific populations may also generate variation in the importance of SC that can drive the evolution of male gonadal investment. In this study, we examined whether variation in population density, which is predicted to influence the level of SC in many animals, is correlated with male gonadal investment among populations of the least killifish, Heterandria formosa, a species with internal fertilization in which multiple mating is common. We complemented this field study by testing whether males respond plastically to experimentally increased levels of SC by increasing investment in testis. This experiment involved two treatments. In the first, we eliminated the potential for sperm competition (NSC) by housing a single male with a single female. In the second, we created a high risk of SC by housing five males with two females. In the field survey, we found significant differences among populations in density and relative testis mass. However, there was no evidence for a correlation between population density and relative testis mass. In our lab experiment, males did not adjust their gonadal investment in response to experiencing different levels of SC for 4 weeks. Our combined results indicate that gonadal investment in male H. formosa is not related to variation in population density.