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The family of Wnt proteins have been implicated in embryogenesis by regulation of cell fate and pattern formation, and also in human carcinogenesis. Wnt10B was previously shown to be involved in breast cancer development. The present study assessed the association of Wnt10B expression in human gastric cancer tissue specimens with clinicopathological data from these patients. Wnt10B expression in the regulation of gastric cancer cell proliferation and migration capacity in vitro was then investigated. The data revealed that Wnt10B mRNA and protein were upregulated in gastric cancer tissue samples and the upregulated Wnt10B mRNA was associated with gastric cancer metastasizing to lymph nodes. Knockdown of Wnt10B expression reduced gastric cancer cell proliferation and migration, as well as expression of a cell proliferation marker Ki67. Knockdown of Wnt10B expression inhibited tumor cell epithelial-mesenchymal transition by upregulation of E-cadherin and downregulation of N-cadherin. In addition, Wnt10B knockdown also suppressed tumor cell stemness by downregulation of octamer-binding transcription factor 4 and Nanog expression. The present data indicated that Wnt10B expression performs an important role in gastric cancer progression in vitro. Therefore, targeting of Wnt10B expression or activity may be investigated as a possible strategy for the control of gastric cancer.