MicroRNA (miRNA) dysregulation has been associated with carcinogenesis in many cancers, including human colorectal cancer (hCRC). However, the effect and mechanism of miR-377-3p on CRC remains elusive. Herein, we first found that miR-377-3p was upregulated in CRC tissues and promoted tumorigenic activity by accelerating the G -S phase transition, promoting cell proliferation and epithelial-mesenchymal transition (EMT) while repressing apoptosis in CRC cells. Glycogen synthase kinase-3β (GSK-3β) was a direct target of miR-377-3p, and upregulated by miR-377-3p. Knockdown of GSK-3β partly rescued miR-377-3p-mediated malignancy characteristics. Most importantly, we showed that miR-377-3p promoted carcinogenesis by activating NF-κB pathway. Taken together, our results first reported that miR-377-3p functions as an oncogene and promotes carcinogenesis via upregulating GSK-3β expression and activating NF-κB pathway in hCRC cells.