Fibroblast growth factor 21 (FGF21), a hormone with multiple metabolic properties, has proven to be pleiotropic biological effects and may play pivotal role in numerous cardiovascular and metabolic diseases in the future. Vascular calcification (VC) is a concomitant pathological process of various cardiovascular and metabolic diseases. However, the effects of FGF21 on VC remain unclear. Therefore, in this research, we aimed to explore the roles and mechanisms of FGF21 in VC induced by vitamin D-3 plus nicotine (VDN) treatment rats. After 28 days VDN treatment, the calcium overload was confirmed by blood pressure, ultrasound imaging, calcium content, ALP activity and aortic pathological characteristics. In terms of FGF21, exogenous FGF21 can ameliorate the elevation of blood pressure, aortic calcification and related injury in VC rats. To investigate the mechanisms of FGF21 on VC, the endoplasmic reticulum stress (ERS) mediated apoptosis pathways were tested. As a method to detect apoptosis, the increased positive TUNEL staining cells were alleviated by FGF21 treatment. Furthermore, exogenous FGF21 can suppress the increased ERS chaperone, GRP78, in the calcified aortas. In the three pathways of ERS mediated apoptosis, we found CHOP pathway and caspase-12 pathway were involved in the treatment of FGF21, but not p-JNK/JNK pathway. Our study proved for the first time that FGF21 can inhibit the progress of VC by alleviating ERS mediated apoptosis in rats. FGF21 might be a new target for preventing and treating VC.