Vascular calcification (VC) is a complex, regulated process involved in many disease entities. So far, there are no treatments to reverse it. Exploring novel strategies to prevent VC is important and necessary for VC-related disease intervention. In this study, we evaluated whether MOTS-c, a novel mitochondria-related 16-aa peptide, can reduce vitamin D3 and nicotine-induced VC in rats. Vitamin D3 plus nicotine-treated rats were injected with MOTS-c at a dose of 5 mg/kg once a day for 4 weeks. Blood pressure, heart rate, and body weight were measured, and echocardiography was performed. The expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and the angiotensin II type 1 (AT-1) and endothelin B (ET-B) receptors was determined by Western blot analysis. Our results showed that MOTS-c treatment significantly attenuated VC. Furthermore, we found that the level of phosphorylated AMPK was increased and the expression levels of the AT-1 and ET-B receptors were decreased after MOTS-c treatment. Our findings provide evidence that MOTS-c may act as an inhibitor of VC by activating the AMPK signaling pathway and suppressing the expression of the AT-1 and ET-B receptors.