To understand the processes that maintain genetic diversity is a long-standing challenge in evolutionary biology, with implications for predicting disease resistance, response to environmental change, and population persistence(1-3). Simple population genetic models are not sufficient to explain the high levels of genetic diversity sometimes observed in ecologically important traits(2). In guppies (Poecilia reticulata), male colour pattern is both diverse and heritable, and is arguably one of the most extreme examples of morphological polymorphism known(4,5). Negative frequency-dependent selection (NFDS), a form of selection in which genotypes are favoured when they are rare(6), can potentially maintain such extensive polymorphism, but few experimental studies have confirmed its operation in nature(7,8). Here we use highly replicated experimental manipulations of natural populations to show that males with rare colour patterns have higher reproductive fitness, demonstrating NFDS mediated by sexual selection. Rare males acquired more mates and sired more offspring compared to common males and, as previously reported, had higher rates of survival(8). Orange colour, implicated in other studies of sexual selection in guppies, did predict male reproductive success, but only in one of three populations. These data support the hypothesis that NFDS maintains diversity in the colour patterns of male guppies through two selective agents, mates and predators. Similar field-based manipulations of genotype frequencies could provide a powerful approach to reveal the underlying ecological and behavioural mechanisms that maintain genetic and phenotypic diversity.