Aim To study the effect of cystamine on the proliferation of human pulmonary arterial smooth muscle cells (PASMC) induced by 5-hydroxytryptamine (5-HT) and RhoA signaling pathway. Methods Human PASMC were cultured and stimulated with 1 μmol/L 5-HT and intervened with cystamine. CCK8 assay was used to observe cell proliferation of PASMC. The protein molecules of RhoA signaling pathway were detected by Western blot and co-immunoprecipitation. Results 5-HT induced the proliferation of PASMC, and increased significantly RhoA serotonylation, RhoA membrane translocation, Rho-associated protein kinase 2 (ROCK2) expression, and the phosphorylations of myosin phosphatase target subunit 1 (MYPT1), protein kinase B (Akt) and extracellular regulated protein kinase (ERK). Cystamine inhibited PASMC proliferation, RhoA serotonylation and the phosphorylations of MYPT1, Akt, ERK induced by 5-HT, but had no significant effect on RhoA membrane translocation and ROCK2 protein expression. Conclusion Cystamine inhibits 5-HT-induced proliferation of human PASMC by inhibiting RhoA serotonylation.