MicroRNAs (miRNAs) have been recognised to regulate cancer development and progression in carcinogenesis as either oncogenes or tumour suppressor genes. However, whether miR-203 plays a crucial role in human laryngeal squamous cell carcinoma (LSCC) remains largely unclear. In the study, we have found that miR-203 expression was significantly lower in LSCC tissues than that in corresponding adjacent non-neoplastic tissues and was negatively correlated with ASAP1 expression level. Lower expression of miR-203 was significantly related to poor differentiation, advanced clinical stages, T3-4 tumour grade, lymph node metastasis and decreased 5-year overall survival. Transfection with miR-203 inhibited proliferation, reduced invasion, induced apoptosis and caused G1 phase cell cycle arrest of Hep-2 cells in vitro, suggesting that miR-203 functioned as a tumour suppressor. We have also tested that over-expression of miR-203 may both suppress the growth of xenograft tumours in mice and downregulate the expressions of ASAP1 in vivo. Furthermore, miR-203 may regulate the expressions of mesenchymal transition (EMT) marker of E-cadherin and cancer stem cells (CSCs) marker of CD44. These findings suggest that miR-203 plays a role as a tumour suppressor in LSCC, likely by regulating ASAP1, probably in relation to EMT and CSCs and may serve as a potential target for therapeutic intervention.