Collagen, as a commercial and functional protein, is widely applied in food, comestic and biomedicine industries, which has a great market demand in recent years. Swim bladder is an ideal optional source for collagen production, due to its high collagen content and low risk of infectious pathogens. Previously, we found that dietary hydroxyproline (Hyp) could promote grow performance and collagen deposition in swim bladder of Nibea coibor, an economic Sciaenidae fish for production of high-grade swim bladder. However, little was known concerning the molecular mechanism underlying the effects of Hyp on collagen deposition. Here, we demonstrated that Hyp could up-regulate collagen I, prolyl hydroxylase (PH) contents and expression levels of genes related to collagen synthesis (col1a1, col1a2, p4ha1, etc.). RNA sequencing indicating that the “Biosynthesis of amino acids” pathway was significantly enriched by Hyp. Correspondingly, the amino acid profile of swim bladder cell was significantly affected by Hyp. Notably, the contents of major substrate amino acids for collagen synthesis (glycine, proline, etc.) were significantly increased, which may be partially related to the up-regulation of shmt gene involving in glycine biosynthesis. Consistent with the results in vitro, the role of Hyp in collagen I deposition, amino acid profile, expression of key regulatory genes in the enriched pathways were further verified in swim bladder of Nibea coibor. These results suggested that dietary Hyp could promote collagen deposition in swim bladder by enhancing collagen synthesis and regulating biosynthesis of amino acids (glycine, proline, etc.) in swim bladder. Our study would provide new insights into the regulatory mechanism underlying the effects of Hyp on collagen deposition in fish and lay foundation for developing nutritional regulation strategy to promote collagen production from aquatic species.