The specific impact of reduced temperature on skeletal muscle adaptation has been poorly investigated. Cold water immersion, one situation leading to decreased skeletal muscle temperature, is commonly proposed to reduce the perception of fatigue and muscle soreness after strenuous exercise. In contrast, it may impair long-term benefits of resistance exercise training on muscle strength and hypertrophy. To date, the physiological factors responsible for this blunted muscle adaptation remain unclear. Here, we used a cell culture model of human primary myotubes to specifically investigate the intrinsic behavior of muscle cells during mild hypothermia (MH). Newly formed myotubes were exposed to either 37°C or 32°C to evaluate the effect of MH on myotube size and morphology, protein synthesis, and anabolic signaling. We also compared the glutamine (GLUT)-induced hypertrophic response between myotubes incubated at 32°C or 37°C. We showed that 48 h exposure to MH altered the cellular morphology (greater myotube area, shorter myosegments, myotubes with irregular shape) and impaired GLUT-induced myotube hypertrophy. Moreover, MH specifically reduced protein synthesis at 8 h. This result may be explained by an altered regulation of ribosome biogenesis, as evidenced by a lower expression of 45S pre-ribosomal RNA and MYC protein, and a lower total RNA concentration. Furthermore, MH blunted GLUT-induced increase in protein synthesis at 8 h, a finding consistent with an impaired activation of the mechanistic target of rapamycin pathway. In conclusion, this study demonstrates that MH impairs the morphology of human myotubes and alters the hypertrophic response to GLUT.