BACKGROUND/OBJECTIVES: Endoplasmic reticulum (ER) stress in adipose tissue causes an inflammatory response and leads to metabolic diseases. However, the association between vitamin D and adipose ER stress remains poorly understood. In this study, we investigated whether 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) alleviates ER stress in adipocytes.MATERIALS/METHODS: 3T3-L1 cells were treated with different concentrations (i.e., 10–100 nM) of 1,25(OH)2 D 3 after or during differentiation (i.e., on day 0–7, 3–7, or 7). They were then incubated with thapsigargin (TG, 500 nM) for an additional 24 h to induce ER stress. Next, we measured the mRNA and protein levels of genes involved in unfold protein response (UPR) and adipogenesis using real-time polymerase chain reaction and western blotting and quantified the secreted protein levels of pro-inflammatory cytokines. Finally, the mRNA levels of UPR pathway genes were measured in adipocytes transfected with siRNA-targeting Vdr. RESULTS: Treatment with 1,25(OH) 2 D 3 during various stages of adipocyte differentiation significantly inhibited ER stress induced by TG. In fully differentiated 3T3-L1 adipocytes, 1,25(OH) 2 D 3 treatment suppressed mRNA levels of Ddit3, sXbp1, and Atf4 and decreased the secretion of monocyte chemoattractant protein-1, interleukin-6, and tumor necrosis factor-α. However, downregulation of the mRNA levels of Ddit3, sXbp1, and Atf4 following 1,25(OH) 2 D 3 administration was not observed in Vdr-knockdown adipocytes. In addition, exposure of 3T3-L1 preadipocytes to 1,25(OH) 2 D 3 inhibited transcription of Ddit3, sXbp1, Atf4, Bip, and Atf6 and reduced the p-alpha subunit of translation initiation factor 2 (eIF2α)/eIF2α and p-protein kinase RNA-like ER kinase (PERK)/PERK protein ratios. Furthermore, 1,25(OH) 2 D 3 treatment before adipocyte differentiation reduced adipogenesis and the mRNA levels of adipogenic genes. CONCLUSIONS Our data suggest that 1,25(OH) 2 D 3 prevents TG-induced ER stress and inflammatory responses in mature adipocytes by downregulating UPR signaling via binding with Vdr. In addition, the inhibition of adipogenesis by vitamin D may contribute to the reduction of ER stress in adipocytes.