Introduction Cardiovascular (CV) diseases (CVD) are the leading cause of mortality worldwide and their impact will increase due to the gradual aging of population. Vascular calcification, which is defined as a formation of hydroxyapatite crystals in the vascular wall, is a serious pathological condition often accompanying CVD and it is a strong predictor of CV events and mortality. Vascular smooth muscle cells (VSMCs) are one of the main type of cells defining development and progression of CVD and vascular calcification. Copper (Cu) is an essential microelement for humans, and it is involved in a plethora of processes affecting CV health. However, copper significance in pathophysiology of VSMCs is critically understudied. Objective The aim was to investigate Cu role in relevant physiological responses of VSMCs. Method The work was carried out on human aortic primary VSMCs. We utilised standard biochemical, molecular and cell biology approaches as well as transcriptome sequencing to assess roles of Cu in VSMCs calcification and migration by means of Cu supplementation and chelation. Results Physiological levels of Cu (10 uM) inhibited calcification of VSMCs, stimulated their metabolic activity and protein synthesis as well as promoted migration. Cu chelation reversed the observed effects and at higher doses was lethal for VSMCs. On the other hand, excessive doses of Cu (up to 150 uM) were also detrimental for VSMCs indicating narrow range of beneficial concentrations. Low micromolar concentrations of Cu inhibited alkaline phosphatase activity both associated with cells and in cell-conditioned media. According to the transcriptomics data, further confirmed by quantitative PCR, physiological amount of Cu during calcification inhibited osteogenic transdifferentiation of VSMCs, reduced cellular immunity-related reactions and promoted synthesis of extracellular matrix (ECM) structural proteins at the initiation stage of the calcification process. Conclusion Summarising, physiological concentration of Cu inhibited in vitro calcification of VSMCs due to prevention of their osteogenic differentiation and stabilisation of ECM. Taken together, our results underline importance of Cu homeostasis for CV health and suggest targeting Cu metabolism in development of new preventive and therapeutical approaches for CVD. IK, JHB and IO were part-funded by the European Union's Horizon 2020 programme (H2020-RISE 2016-734931, MILEAGE Project) and IO was also supported by a Vernadski PhD scholarship from the French government.