MicroRNA deregulation is involved in tumor initiation and progression. The aim of this study was to identify and validate the microRNA candidates that contribute to the metastasis of oral tongue squamous cell carcinoma (OTSCC). Using microarrays, a panel of differentially expressed microRNAs was identified in paired OTSCC cell lines with different metastatic potential. Selected microRNA candidates (including hsa-miR-222) were further validated using quantitative PCR approach. Functional analysis indicated that hsa-miR-222 inhibits OTSCC cell invasion. Ectopic transfection of hsa-miR-222 reduced the expression of MMP1 and SOD2 in OTSCC cell lines. Direct targeting of hsa-miR-222 to specific sequences located in the 3'-untranslated regions of both MMP1 and SOD2 mRNAs were confirmed using luciferase reporter gene assays. Furthermore, SOD2 knockdown by siRNA led to the downregulation of MMP1 expression. Taken together, these results suggested that hsa-miR-222 regulates the MMP1 expression through both direct cis-regulatory mechanism (targeting MMP1 mRNA) and indirect trans-regulatory mechanism (indirect controlling of MMP1 gene expression by targeting SOD2). Our results indicate that hsa-miR-222 plays an important role in OTSCC invasion, and may serve as a novel therapeutic target for OTSCC patients at risk of metastatic disease.