Objective: To explore the regulatory mechanism of miR-205-3p targeting Laminin gamma 2-chain (LAMC2) gene on the biological characteristics of oral stem squamous cell carcinoma (OSCC) stem cells. Methods: The targeting relationship between miR-205-3p and LAMC2 gene was predicted by the bioinformatic database and verified by dual luciferase reporter assay system. qRT-PCR was used to detect the expression of miR-205-3p and LAMC2 in epithelial tissues from patients with OSCC or benign mass in oral mucosa. Normal oral mucosa epithelial cells were selected as the normal group. OSCC stem cells were divided into blank group, miR-205-3p mimic group, miR-205-3p inhibitor group, siRNA-LAMC2 group and miR-205-3p mimic + siRNA-LAMC2 group, respectively. qRT PCR and western blot were performed to investigate the expression level of miR-205-3p, LAMC2, Bax, Bcl-2 and epithelial-mesenchymal transition (EMT)-related factors in cells of each group. Cell proliferation, invasion ability and apoptosis were detected by MTT assay, transwell assay and flow cytometry, respectively. Results: Compared with tissues from patients with benign mass of oral mucosa, reduced miR-205-3p expression level and enhanced LAMC2 expression level were detected in epithelial tissue from OSCC patients (both P<0.05). Compared with the blank group, up regulation of miR-205-3p or down-regulation of LAMC2 could promote expression levels of E-cadherin and Bax, and suppress expression levels of N-cadherin, vimentin, and Bcl-2 in OSCC stem cells, which could also inhibit proliferation, invasion ability and facilitate apoptosis of OSCC stem cells (all P<0.05). In the miR-205-3p mimic + siRNA-LAMC2 group, the changes of above indicators were more notable. Inhibition of the miR-205-3p expression had the opposite effect (all P<0.05). Conclusion: Up regulation of miR-205-3p can inhibit proliferation, invasion ability and promote apoptosis of OSCC stem cells by inhibiting LAMC2 gene expression.