Our study aimed to evaluate the dualistic effect of heat stress on muscle differentiation at different temperatures, and whether vitamin E, a powerful antioxidant, could offset any negative effects, using bovine skeletal-muscle-derived cells (BSMCs) with myogenic properties. The BSMCs were extracted from the skeletal muscle of thirty-month-old Korean native cattle and subjected to myogenic differentiation under three heat exposure conditions: 37°C (control; CON), 39°C (mild heat stress; MHS), and 41°C (severe heat stress; SHS) for 24h with or without vitamin E treatment (NE or VE). After 24h treatments, the cells were returned to 37°C incubators and differentiated until day 6. On day 1, because of the heat exposure, the gene expression of MYOG was the highest in MHS (P = 0.047), suggesting a promotive effect of mild heat stress on myogenic differentiation, while on day 6, compared to CON and MHS, MYOD (P = 0.013) and MYOG (P = 0.029) were the lowest in SHS. Vitamin E treatment also lowered MYOG (P = 0.097), regardless of heat exposure. On day 1, HSPB1 (P = 0.001) and HSP70 (P < 0.001) were the highest in SHS, and an interaction between heat exposure and vitamin E treatment was found on day 6 (P < 0.027). BCL-2 was also the highest on day 1 in SHS (P = 0.05), and an interaction of heat exposure and vitamin E treatment was found on day 1 on BAX expression (P = 0.038). For antioxidant genes, SOD1 (P = 0.002) and GPX1 (P < 0.001) were affected by heat exposure, with the highest levels being observed in SHS, and on day 6, GPX1 was still the highest in SHS (P = 0.027). The fusion index was also affected by heat exposure, showing a decrease in SHS and an increase in MHS compared to CON (P < 0.001). Significant effects were noted from heat exposure (P < 0.001), vitamin E treatment (P < 0.001), and the interaction of heat exposure and vitamin E treatment (P = 0.002) on the protein content. Taken together, our findings provide evidence that vitamin E could ameliorate the harmful effects of heat exposure by modulating heat shock proteins and apoptosis regulators, improving the protein synthesis of BSMCs during myogenic differentiation. These results suggest that vitamin E supplementation could potentially protect muscle development in beef cattle under summer heat stress.