Under fasting conditions, the pathway converting gluconeogenesis precursors into muscle glycogen becomes crucial due to reduced glycogen reserves. However, there is limited research on skeletal muscle gluconeogenesis and the impact of fasting on gluconeogenic gene expression. Sheep fetal skeletal muscle cells cultured in vitro were used to study the effects of varying lactic acid concentrations (0 to 30 mM) and 2.5 mM glucose on the expression of gluconeogenesis-related genes after 6 h of fasting. The effects on mRNA and protein expression of key genes involved in skeletal muscle gluconeogenesis were measured by quantitative real time polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting at 48 h. Fasting increased the expression of key gluconeogenic genes, fructose-1,6-bisphosphatase 2 (FBP2), glucose-6-phosphatase 3 (G6PC3), pyruvate kinase M (PKM), monocarboxylate transporter1 (MCTS1), glucose transporter type 4 (GLUT4), pyruvate carboxylase (PC), and lactate dehydrogenase A (LDHA). The mRNA levels of FBP2, G6PC3, and MCTS1 significantly decreased with glucose addition. Additionally, 10 mM lactic acid significantly promoted the expression of FBP2, PC, MCTS1, LDHA, GLUT4, and PKM while inhibiting phosphoenolpyruvate carboxykinase (PEPCK) expression. At the protein level, 10 mM lactic acid significantly increased FBP2 and PKM protein expression. This study shows that fasting regulates key gluconeogenic gene expression in sheep skeletal muscle cells and highlights the role of lactic acid in inducing these gene expressions.