Low temperature is an important environmental factor affecting the breeding industry. Due to sudden cold waves or seasonal changes, huge losses have been caused to the breeding industry all over the world. We investigated the effect of temperature gradient (25, 19, and 13 °C) and time (0, 6, 24, and 96 h) on the histomorphology, antioxidant enzyme activity, mitogen-activated protein kinase pathway, and lipidomics of the skeletal muscle from T. fasciatus. The results showed that low temperature damaged muscle microstructure and caused antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase and catalase) upregulation. The mitogen-activated protein kinase (MAPK), including extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and p38MAPK, was upregulated under cold stress. The expression of genes involved in lipogenesis [6-phosphate glucose dehydrogenase, glucose-6-phosphate dehydrogenase, fatty acid synthase, acetyl-coa-carboxylase, lipoprteinlipase, sterol-regulatory element binding protein-1], lipolysis [hormone-sensitive lipase, and carnitine palmitoyltransferase-1], and transcription [peroxisome proliferator-activated receptor α, peroxisome proliferator-activated receptor γ] were upregulated. Lipidomics results showed that phospholipid metabolites increased under cold stress, which compensated for the integrity of cell membranes. Overall, this study showed that T. fasciatus may adjust fat metabolism through MAPK to compensate for cell membrane damage in muscles caused by low temperature. Our results reveal the possibility that phospholipid metabolites can be used as potential feeds, and further reveal the mechanism of resistance to low temperatures.