Although genetic diversity within a population is suggested to improve population-level fitness and productivity, the existence of these effects is controversial because empirical evidence for an ecological effect of genetic diversity and the underlying mechanisms is scarce and incomplete. Here, we show that the natural single-gene behavioural polymorphism (Rover and sitter) in Drosophila melanogaster has a positive effect on population fitness. Our simple numerical model predicted that the fitness of a polymorphic population would be higher than that expected with two monomorphic populations, but only under balancing selection. Moreover, this positive diversity effect of genetic polymorphism was attributable to complementarity effect, rather than to selection effect. Our empirical tests using the behavioural polymorphism in D. melanogaster clearly supported the model predictions. These results provide direct evidence for an ecological effect of genetic diversity on population fitness and its condition dependence.