Abstract: Swim bladder is an ideal optional source for collagen production and has long been considered as a traditional tonic in China. Therefore, strategies are required improve collagen content in the swim bladder for producing products with high nutrition and economic value. Proline (Pro) is the main proteinogenic amino acid needed for collagen production. Our previous study found that Pro can promote collagen deposition in the swim bladder of Chu’s croaker (Nibea coibor), and it is potentially regulated by TGF-β/Smad signaling pathway. In order to further verify the role of TGF-β/Smad signaling pathway in Pro promoting collagen deposition in swim bladder, the content collagen and transcription levels of related genes in the swim bladder of N. coibor were analyzed by inhibiting the TGF-β/Smad pathway. It was demonstrated that proline improved significantly the collagen content in the swim bladder of N. coibor, although no obvious enhancement in growth performance was observed. Proline supplementation up-regulated genes involved in collagen biosynthesis in the swim bladder. However, oxymatrine interference reduced significantly collagen deposition and the expression of Col1α1, Col1α2, TGF-β and Smad2 genes in the swim bladder of N. coibor. These results suggested that proline promoted collagen deposition in swim bladder mainly through regulating TGF-β/Smad signaling pathway, most likely via transient overexpression of Col1α1, Col1α2, TGF-β and Smad2 genes. Our results help to understand the molecular mechanism of collagen deposition in N. coibor for developing new strategies of producing fish products with higher quality.