Prolactin is a hormone conserved across all vertebrates and is renowned for its role in reproduction and parental care. Previous studies on prolactin in fish have primarily relied on administration of mammalian prolactin and have suggested that increases in prolactin lead to greater parental care. However, the influence of endogenous prolactin on fish parental care remains unknown. Here, we measure circulating concentrations of endogenous prolactin during parental care in a fish and link these concentrations to parental care behaviour. We provide evidence that male bluegill sunfish with higher circulating concentrations of prolactin provide more parental care to their offspring. Furthermore, we show that nesting males with experimentally reduced perceived paternity have lower circulating prolactin concentrations and perform fewer parental behaviours, facilitating an adaptive investment in offspring in response to paternity cues. Our findings not only confirm the role of endogenous prolactin in modulating parental care behaviour in a fish but also provide a mechanism underlying the adaptive changes in parental care made in response to perceived paternity.