Chronic hypoxia-induced pulmonary arterial hypertension (HPH) is closely associated with profound vascular remodeling, especially pulmonary arterial medial hypertrophy and muscularization due to hyperplasia of pulmonary artery smooth muscle cells (PASMCs). Aberrant Wnt signaling has been associated with lung diseases, but its role in pulmonary hypertension is unclear. This study evaluated the effect of Wnt5a on hypoxia-induced proliferation of human PASMCs and its possible mechanism. The results show that hypoxia (3% O(2), 48 h) induced proliferation of human PASMCs, accompanied with a significant decrease in Wnt5a gene expression, increase in β-catenin and Cyclin D1 expression, as well as β-catenin nuclear translocation. Treatment with recombinant mouse Wnt5a significantly inhibited hypoxia-induced proliferation of human PASMCs, upregulation of Cyclin D1 and β-catenin expression, as well as the nuclear translocation of β-catenin. These effects were inhibited by Wnt5a antibody. Knocking down β-catenin or Cyclin D1 gene expression inhibited hypoxia-induced human PASMC proliferation, whereas overexpression of β-catenin increased hypoxia-induced human PASMC proliferation and counteracted the inhibitory effect of Wnt5a. These results suggest that Wnt5a has an antiproliferative effect on hypoxia-induced human PASMC proliferation by downregulation of β-catenin and its target gene Cyclin D1. Hypoxia-induced downregulation of Wnt5a may be a way to facilitate hypoxia-induced human PASMC proliferation. The results of this study will help to understand the novel strategies for PH treatment involving Wnt signaling.