Background/Objectives: Myogenesis, the process by which myoblasts differentiate into multinucleated muscle fibers, is tightly regulated by transcription factors, signaling pathways, and metabolic cues. Among these, fatty acids have emerged as key regulators beyond their traditional role as energy substrates. Oleic acid, a monounsaturated fatty acid, has been shown to modulate muscle differentiation, potentially influencing myogenic pathways. This study examines the role of oleic acid in promoting C2C12 myoblast differentiation and its associated molecular mechanisms, comparing it to standard horse serum (HS)-based differentiation protocols. Methods: C2C12 murine myoblasts were cultured under proliferative conditions and differentiated using DMEM supplemented with either 2% HS or oleic acid (C18:1, n-9). The molecular signaling pathway was evaluated by measuring the expression of p38 MAPK, β-catenin, GLUT4, and NDRG1. Results: Oleic acid promoted the differentiation of C2C12 cells, as evidenced by a progressively elongated morphology, as well as the induction of muscle-specific myogenin, myosin heavy chain (MHC), and MyoD. Moreover, oleic acid reduced the expression of Atrogin-1 and MuRF1 ubiquitin E3 ligase. BODIPY staining revealed the enhanced accumulation of lipid droplets in oleic acid-treated cells. The Western blot analysis demonstrated robust activation of p38 MAPK and β-catenin pathways in response to oleic acid, compared with HS. Additionally, oleic acid upregulated GLUT4 expression and increased the phosphorylation of insulin receptor and NDRG1, indicating an enhanced glucose uptake capacity. Conclusions: These findings demonstrate that oleic acid promotes C2C12 myoblast differentiation and improves glucose uptake via GLUT4. Oleic acid emerges as a promising metabolic regulator of myogenesis, offering potential therapeutic applications for muscle regeneration in muscle-related pathologies.