MicroRNA deregulation is a critical event in tumor initiation and progression. The down-regulation of microRNA-138 has been frequently observed in various cancers, including tongue squamous cell carcinoma (TSCC). Our previous studies suggest that deregulation of miR-138 is associated with the enhanced proliferation and invasion in TSCC cells. Here, we seek to identify the targets of miR-138 in TSCC, and explore their functional relevance in tumorigenesis. Our genome-wide expression profiling experiments identified a panel of 194 unique transcripts that were significantly down-regulated in TSCC cells transfected with miR-138. A comprehensive screening using six different sequence-based microRNA target prediction algorithms revealed that 51 out of these 194 down-regulated transcripts are potential direct targets for miR-138. These targets include: chloride channel, nucleotide-sensitive, 1A (CLNS1A), G protein alpha inhibiting activity polypeptide 2 (GNAI2), solute carrier family 20, member 1 (SLC20A1), eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), and Rho-related GTP-binding protein C (RhoC). GNAI2 is a known proto-oncogene that is involved in the initiation and progression of several different types of tumors. Direct targeting of miR-138 to two candidate binding sequences located in the 3'-untranslated region of GNAI2 mRNA was confirmed using luciferase reporter gene assays. Knockdown of miR-138 in TSCC cells enhanced the expression of GNAI2 at both mRNA and protein levels. In contrast, ectopic transfection of miR-138 reduced the expression of GNAI2, which, in consequence, led to reduced proliferation, cell cycle arrest and apoptosis. In summary, we identified a number of high-confident miR-138 target genes, including proto-oncogene GNAI2, which may play an important role in TSCC initiation and progression.