Aim To study the effect of fluoxetine on serotonin-induced RhoA signalling pathway in pulmonary arterial smooth muscle cells.Methods The pulmonary arterial smooth muscle cells of rat were exposed to serotonin for 48 h with or without fluoxetine or fasudil. Then pulmonary arterial smooth muscle cells proliferation was tested by 3-［4,5-dimethylthylthiazol-2-yl］-2,5 diphenyltetrazolium broide assay. The cellular extracts were prepared for coimmunoprecipitation or Western blot of RhoA signalling pathway.Results Serotonin promoted pulmonary arterial smooth muscle cells proliferation, increased RhoA serotonylation, RhoA membrane translocation, Rho kinase 2 protein expression and myosin phosphatase target subunit 1, extracellular regulated protein kinase and protein kinase B phosphorylation. Fluoxetine inhibited these changes dose-dependently. However, fasudil did not inhibit increased RhoA serotonylation induced by serotonin.Conclusion Fluxetine inhibited serotonin-induced pulmonary arterial smooth muscle cells proliferation via inhibition of RhoA signalling pathway.