Vascular calcification (VC) is a major risk factor for increasing cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Indoxyl sulfate (IS), a representative uremic toxin, is closely associated with VC in CKD patients. Matrix Gla protein (MGP) plays pivotal role in VC as a calcification inhibitor. The aim of this work was to explore whether MGP was involved in IS-induced VC. Here, we demonstrated the role of MGP in the IS-induced osteogenic differentiation of human aortic smooth muscle cells (HASMCs). The methods included Von Kossa staining, immunohistochemistry, Alizarin Red staining, quantitative real-time PCR and western blotting. MGP was decreased in calcified arteries both in CKD patients and rats. In vitro, IS suppressed MGP expression in HASMCs by activating ROS/NF-κB signaling in parallel with osteogenic differentiation, which was mitigated by inhibiting ROS and NF-κB with diphenyleneiodonium and Bay11-7082. Further investigation showed that IS induced NF-κB-responsive microRNA (miR)-155-5p mediating MGP downregulation. Overexpression of miR-155-5p with mimics aggravated IS-induced MGP reduction and osteogenic differentiation. In contrast, these conditions were diminished by silencing miR-155-5p. We demonstrate that IS promotes the HASMCs phenotype switch by suppressing MGP expression via ROS/NF-κB/miR-155-5p signaling and provide a new insight for the pathogenesis of IS-induced VC. •MGP expression is decreased in medial calcified vessels of CKD patients and rats.•Downregulation of MGP occurs in IS-induced osteogenic differentiation of HASMCs.•IS suppressed MGP expression by activating ROS/NF-κB/miR-155-5p signaling pathway.