Objective: To investigate the relationship between the expression of Golgi phosphoprotein 3 (GOLPH3) gene and the activity of Wnt signaling pathway in human colon cancer cells. Methods: The GOLPH3 mRNA expressions in 4 different human colon cancer cell lines (HCT116, HT29, SW480 and SW620) were detected by RT-PCR, after which, the cell line with highest GOLPH3 mRNA expression was selected and then underwent GOLPH3 gene interference, and the RNA interference effect was examined by RT-PCR. In the colon cancer cells after interference, the changes in the activity of Wnt signaling pathway, proliferative ability and protein expressions of GOLPH3 and β-catenin were detected by TOPFlash reporter gene assay, flat plate colony-forming assay and Western blot analysis, respectively. Results: SW620 cells showed the highest relative GOLPH3 mRNA expression among the 4 types of colon cancer cell lines. The relative GOLPH3 mRNA expression was significantly reduced after GOLPH3 siRNA transfection in SW620 cells (P<0.001). In SW620 cells after GOLPH3 siRNA transfection compared with untreated SW620 cells, the transcriptional activity of Wnt signaling pathway was significantly decreased (0.342 vs. 1.000, P<0.001), the number of cancer cell colon formation was significantly reduced (82.333 vs. 207.333, P<0.001), and both GOLPH3 and β-catenin protein expressions were significantly down-regulated (0.260 vs. 1.00; 0.182 vs. 1.00, both P<0.001). Conclusion: In human colon cancer cells, increased GOLPH3 expression may up-regulate the acitivity of Wnt/β-catenin signaling pathway, and thereby promote cell proliferation.