Aim To explore whether 1,5-(OH)₂D₃ can regulate the autophagy and calcification of human umbilical vein endothelial cells (HUVEC) and the calcification of mouse atherosclerotic plaque through Ca²⁺/CaM signal. Methods (1) HUVEC were incubated in high fat microenvironment to stimulate autophagy. The autophagy level was detected by Western blot, fluorescence microscopy, transmission electron microscopy after HUVEC being stimulated by 1,5-(OH)₂D₃. Lentivious were used to overexpress or silence calmodulin (CaM), and the calcification was tested by Western blot,calcium ion detection kit, Alizarin red. (2) ApoE－/－ mice fed with high fat diet were randomly divided into 3 groups:control group (1,5-(OH)₂D₃ 2.5 μg/(kg·d) dissolved in 0.1 mL soybean oil, Gavage)； CaM overexpressed group (control +CaM overexpressed lentivirus, Caudal vein injection) and CaM silenced group (control+CaM silenced lentivirus, Caudal vein injection). Scanning electron microscope was used to observe autophagosomes and calcification of the aorta. HE and Von Kossa staining were used to detect the As degree and calcification of the aorta, respectively. Results The autophagy was promted and the expression of Ca²⁺ and CaM were up-regulated after HUVEC being stimulated by 1,5-(OH)₂D₃. Compared with the control group, CaM overexpressed group showed enhanced autophagy and reduced calcification in HUVEC and As plaque in ApoE－/－mice. While the CaM silencing group showed autophagy dysfunction, increased calcium depositions in HUVEC and plaques. Conclusion 1,5-(OH)₂D₃ regulates HUVEC autophagy to inhibit cell calcification and plaque calcification through Ca²⁺/CaM signaling.