To study the effects of CTNNB1 gene knockout by CRISPR-Cas9 technology on cell adhesion, proliferation, apoptosis, and Wnt/beta-catenin signaling pathway. CTNNB1 gene of HEK 293T cells was knocked out by CRISPR-Cas9. This was confirmed by sequencing and western blotting. Methylthiazolyl-tetrazolium bromide assays indicated that deletion of beta-catenin significantly weakened adhesion ability and inhibited proliferation rate (P < 0.01) of HEK 293T cells. Nevertheless, deletion of beta-catenin did not affect apoptosis of HEK 293T cells, which was analyzed by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide double staining. In addition, expression level of GSK-3 beta, CCND1, and CCNE1 detected by qPCR and expression level of N-Cadherin and cyclin D1 detected by western blotting were significantly decreased (P < 0.01) while expression of gamma-catenin detected by western blotting was significantly increased (P < 0.001). Knockout of CTNNB1 disturbed Wnt/beta-catenin signaling pathway and significantly inhibited adhesion and proliferation of HEK 293T cells.