Heightened expression of the S100 calcium–binding protein, S100A4/Mts1, is observed in pulmonary vascular disease. Loss of serotonin (5-hydroxytryptamine [5-HT]) receptors or of the serotonin transporter (SERT) attenuates pulmonary hypertension in animals, and polymorphisms causing gain of SERT function are linked to clinical pulmonary vascular disease. Because 5-HT induces release of S100β, we investigated the codependence of 5-HT receptors and SERT in regulating S100A4/Mts1 in human pulmonary artery smooth muscle cells (hPA-SMC). 5-HT elevated S100A4/Mts1 mRNA levels and increased S100A4/Mts1 protein in hPA-SMC lysates and culture media. S100A4/Mts1 in the culture media stimulated proliferation and migration of hPA-SMC in a manner dependent on the receptor for advanced glycation end products. Treatment with SB224289 (selective antagonist of 5-HT_1B), fluoxetine (SERT inhibitor), SERT RNA-interference, and iproniazid (monoamine oxidase-A inhibitor), blocked 5-HT–induced S100A4/Mts1. 5-HT signaling mediated phosphorylation (p) of extracellular signal–regulated kinase 1/2 (pERK1/2), but pERK1/2 nuclear translocation depended on SERT, monoamine oxidase activity, and reactive oxygen species. Nuclear translocation of pERK1/2 was required for pGATA-4–mediated transcription of S100A4/Mts1. These data provide evidence for a mechanistic link between the 5-HT pathway and S100A4/Mts1 in pulmonary hypertension and explain how the 5-HT_1B receptor and SERT are codependent in regulating S100A4/Mts1.