Aim To study the role of endoplasmic reticulum stress (ERS) in the inhibition of vascular calcification by fibroblast growth factor 21 (FGF21) in rats. Methods Adult male SD rats were randomly divided into control group, model group, FGF21 group and FGF21＋tunicamycin (TM) group. Vitamin D3 combined with nicotine was used to establish vascular calcification model in the latter 3 groups. FGF21 group was intraperitoneally injected with 1 mg/(kg·d) of FGF21, and FGF21＋TM group was intraperitoneally injected with 1 mg/(kg·d) of FGF21 and 4.5 mg/(kg·w) of TM, for 28 consecutive days. Alizarin red staining, calcium content, alkaline phosphatase (ALP) activity, TUNEL staining, osteogenesis marker gene and ERS gene expression levels in thoracic aorta of rats were compared among groups. Results Alizarin red staining of thoracic aorta in FGF21 group was significantly weakened. Calcium content, ALP activity, positive rate of TUNEL staining and protein expression levels of Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), BMP4, osteoprotegerin (OPG), glucose-regulated protein 78 (GRP78), CCAAT/EBP homologous protein (CHOP) and cysteinyl aspartate specific proteinase-12 (Caspase-12) in the FGF21 group were significantly lower than those in the model group (P＜0.05). Positive rate of TUNEL staining and protein expression levels of RUNX2, BMP2, BMP4, OPG, GRP78, CHOP and Caspase-12 in the FGF21＋TM group were significantly higher than those in the FGF21 group (P＜0.05). Conclusion FGF21 can inhibit vascular calcification induced by vitamin D3 and nicotine, and blocking ERS-mediated cell apoptosis and osteogenic differentiation may be its molecular mechanism of inhibiting vascular calcification.