Abstract: Hardness is widely regarded as a critical factor influencing the whole texture of fish flesh. The objective of this study was to elucidate the regulatory mechanism underlying muscle hardness in hybrid bream (BBTB, Megalobrama amblycephala ♀ × Culter alburnus ♂). A comparison of the physiological features of high hardness (HH) and low hardness (LH) muscle revealed that the former had higher contents of collagen Ⅰ and lower muscle fiber diameter. Transcriptomic data revealed that the myofiber assembly pathway and the HIF-1 signaling pathway were activated in HH muscle. At the metabolic level, the categories of amino acids and lipids were the principal differentially abundant metabolites between the HH and LH muscle. The detection of amino acid profiles further revealed significant differences in amino acid metabolism between the HH and LH muscles, with the HH muscle having higher levels of amino acids than the LH muscle, especially hydroxyproline (Hyp). Furthermore, through supplementation of Hyp in BBTB myoblasts, the results indicated that 0.8 mmol/L Hyp increased the proliferation, differentiation, migration, and collagen synthesis of myoblasts. Finally, BBTB was treated with Hyp intraperitoneally for 15 days. The results revealed that 0.1 g/kg Hyp significantly increased muscle hardness, myofiber density, myofibrillar protein synthesis, and HIF-1 protein content. The results obtained in this study indicated that Hyp supplementation promoted collagen synthesis and proliferation of myoblast and muscle fibers in the BBTB, which may be induced by activation of the transcription factor HIF1 and contributes to the impacts of Hyp on improvements in muscle hardness in the BBTB.