We previously showed that brown (pre)adipocytes express Trpv1, a capsaicin receptor, and that capsaicin stimulates differentiation of brown preadipocytes in the late stages of brown adipogenesis. The present study revealed that treatment with 100 μM capsaicin stimulates brown adipogenesis by inducing endoplasmic reticulum (ER) stress. Treatment with capsaicin (100 μM) during brown adipogenesis enhanced lipid accumulation and the expression of Ucp1, a gene selectively expressed in brown adipocytes. Capsaicin treatment also caused an increase in the cytosolic calcium concentration even when extracellular calcium was removed. I-RTX, a Trpv1 inhibitor, did not modulate the increase in cytosolic calcium concentration, lipid accumulation or Ucp1 expression. Previous studies revealed that the release of calcium from the ER induces ER stress, leading to the conversion of X-box binding protein 1 (Xbp1) pre-mRNA to spliced Xbp1 (sXbp1) as well as the up-regulation of Chop expression. Capsaicin treatment increased the expression of sXbp1 and Chop in brown preadipocytes and did not enhance lipid accumulation or Ucp1 expression in Xbp1 knockdown cells. The present results describe a novel mechanism of brown adipogenesis regulation via ER stress that is induced by a supra-pharmacological concentration of capsaicin.