Vascular calcification is the formation of mineralised tissue within the walls of arteries. The pathology has many similarities to embryonic bone formation and involves the osteogenic differentiation of vascular smooth muscle cells (VSMCs) and matrix mineralisation. Recent studies have demonstrated that the bioactive sphingolipids, ceramide and sphingosine-1-phosphate (S1P), regulate embryonic bone formation. Ceramide can be generated by lysosomal acid sphingomyelinase (L-SMase) and neutral sphingomyelinase (N-SMase), and be converted to sphingosine by acid ceramidase (ACDase) and subsequently to S1P by sphingosine kinases (SK1 & SK2). This study tested the hypothesis that ceramide and S1P also regulate VSMC matrix mineralisation.VSMCs were cultured in the presence of 3 mM β-glycerophosphate (BGP) to induce osteogenic differentiation and matrix mineralisation. During VSMC mineralisation there were decreases in the activities of L-SMase and N-SMase and increases in the levels of C18 and C20 ceramide. S1P levels also increased during mineralisation as did SK1 and SK2 mRNA and SK activity. These results demonstrate that ceramide and S1P have the potential to regulate VSMC mineralisation.The exogenous addition of C2 ceramide decreased the rate of VSMC matrix mineralisation. Consistent with this, when VSMCs were cultured with 3 mM BGP and the joint L-SMase and ACDase inhibitor, desipramine, total ceramide levels increased and no matrix mineralisation was detected. These findings suggest that ceramide is an inhibitor of VSMCs matrix mineralisation. It was also noted in the presence of 3 mM BGP and desipramine that the mineralisation-associated increase in S1P was inhibited. In agreement with this, when exogenous S1P was added to the VSMCs an increase in matrix mineralisation was observed. Thus, S1P acts as a promoter of matrix mineralisation.To determine how S1P was promoting matrix mineralisation the signalling roles of the ezrin, radixin and moesin (ERM) proteins were investigated. The short-term stimulation of VSMCs with S1P led to the phosphorylation of the ERM proteins and over the mineralisation time-course, when S1P levels increased, the levels of ERM phosphorylation also increased. When VSMCs were cultured in the presence of 3 mM BGP and the inhibitor of ezrin phosphorylation, NSC668394, a decrease in matrix mineralisation was observed. No increases in ERM phosphorylation were seen in the presence of desipramine during the mineralisation time-course Therefore, S1P may be increasing matrix mineralisation through promoting the phosphorylation of the ERM proteins.This work has demonstrated that ceramide inhibits and S1P promotes VSMC matrix mineralisation in vitro. Additionally, this work identifies activation of ERM proteins, downstream of S1P, as a novel signalling pathway promoting matrix mineralisation. Characterisation of novel regulators of VSMC matrix mineralisation in vitro gives insight into the complex mechanisms contributing to vascular calcification in vivo and will aid in identification of novel therapeutic targets.