This study investigated the effects of microRNA-135a (miR-135a) targeting of glycogen synthase kinase 3β (GSK3β) on the epithelial-mesenchymal transition (EMT), migration and invasion of bladder cancer (BC) cells by mediating the Wnt/β-catenin signaling pathway. BC and adjacent normal tissues were collected from 165 BC patients. Western blotting and quantitative real-time PCR were used to detect the expression of GSK3β, β-catenin, cyclinD1, E-cadherin, vimentin and miR-135a in BC tissues and cells. Cells were assigned to blank, negative control (NC), miR-135a mimics, miR-135a inhibitors, small interfering RNA (siRNA)-GSK3β or miR-135a inhibitors+siRNA-GSK3β groups. miR-135a, β-catenin, cyclinD1 and vimentin expression increased, while GSK3β and E-cadherin expression decreased in BC tissues compared with adjacent normal tissues. Compared with the blank and NC groups, the expression of miR-135a, β-catenin, cyclinD1 and vimentin was higher, and cell proliferation, migration, invasion and tumor growth were increased in the miR-135a mimics and siRNA-GSK3β groups. These groups showed an opposite trend in GSK3β and E-cadherin expression and cell apoptosis. The miR-135a inhibitors group was inversely correlated with the blank and NC groups. It was concluded that miR-135a accelerates the EMT, invasion and migration of BC cells by activating the Wnt/β-catenin signaling pathway through the downregulation of GSK3β expression.