The market demand of collagen has been increasing over years as its wide application in food, comestic and biomedicine industries. Swim bladder is an ideal optional source for collagen production and has long been considered as a traditional tonic in China. Therefore, strategies to improve collagen content in the swim bladder would confer to producing products with high nutrition and economic value. However, little was known in the regulatory mechanism on collagen deposition in fish, especially in the swim bladder. Here, we reported an in-vivo and in-vitro study of proline's effects on collagen deposition in a famous economic marine fish, Chu's croaker (Nibea coibor) for the first time. It was demonstrated that proline significantly improved collagen content in the swim bladder of N. coibor, although no obvious enhancement in growth performance was observed. By RNA-sequencing and qRT-PCR, several genes involved in collagen biosynthesis (Col1a1, Col1a2 and CTGF) and degradation (TIMP2) were identified and validated to be up-regulated in the swim bladder of fish fed with proline supplementation. Furthermore, the effects of proline on collagen deposition and related gene expression were confirmed in swim bladder cell of N. coibor. These results suggested that proline promoted collagen deposition in swim bladder mainly through enhancing collagen biosynthesis and inhibiting collagen degradation. Our data would help to understand the molecular mechanism of collagen deposition in the species and develop new strategies of producing fish products with higher quality.