Eccrine sweat glands play a vital role in human thermoregulation; however, their self-repair function is minimal. Therefore, developing methods to regenerate and improve sweat gland function that use cultured sweat gland cells presents an urgent issue. The tissue microenvironment, especially hypoxic niches, essentially maintain cell stemness, highlighting the importance of oxygen concentration in the culture environment. Therefore, we evaluated the effects of different oxygen environments on human sweat glands and their regulatory mechanisms. Human eccrine sweat glands express HIF-1α and HIF-2α, suggesting that they respond to hypoxia in vivo. Primary human-derived eccrine sweat gland cells were cultured for two weeks using the spheroid culture method at 0.5%, 2%, 10%, and 21% O concentration. HIF-1, Wnt/β-Catenin, and TGFβ1 signaling increased in sweat gland cells cultured in 0.5% O conditions, along with increased undifferentiated cell marker expression. The results of this study will contribute to in vitro research models of sweat glands and treatment development for damage to sweat glands, including burns.