We determined the effect of indoxyl sulfate (IS) on Pit-1 expression and the role of Pit-1 in IS-induced osteoblastic differentiation and calcification of vascular smooth muscle cells (VSMCs). To assess osteoblastic differentiation and Pit-1 expression, VSMCs were incubated with various concentrations of IS for different durations. Phosphonoformic acid (PFA), a competitive inhibitor of Pit-1, was used to verify the role of Pit-1. Western blot analysis and quantitative real-time polymerase chain reaction (PCR) were performed to assess Pit-1 protein and mRNA levels, respectively. To evaluate calcification, calcium content was measured. After IS treatment, we observed osteoblastic differentiation and calcification of VSMCs and up-regulation of Pit-1 expression. Moreover, the effect of IS on osteoblastic differentiation and Pit-1 expression was partly dose- and time-dependent. PFA abrogated the IS-induced osteoblastic differentiation and calcification of VSMCs to a certain extent. The c-Jun N-terminal kinase (JNK) pathway was activated after treatment with IS, whereas inhibition of the JNK pathway partially attenuated the effect of IS on both the stimulation of Pit-1 expression and calcium deposition. Our study is the first to demonstrate that IS promotes Pit-1 expression in part by activation of the JNK pathway that is involved in the mechanism of IS-induced osteoblastic differentiation and matrix mineralization.