Tongue squamous cell carcinoma (TSCC) is a major subtype of head and neck squamous cell carcinoma (HNSCC), which is an intractable cancer with a poor prognosis. Studies have shown that microRNAs (miRNAs) play an important role in TSCC biology. However, the expression and functions of miRNAs in TSCC remain unclear. The non-coding RNA profiles of TSCC were downloaded from the GEO database. WGCNA (Weighted gene co-expression network analysis) and differential expression miRNA (DE-miRNA) analyses were employed to identify key candidate miRNAs. miRNA expression was detected using RT-qPCR analysis. The target genes of key miRNAs were predicted. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to explore the potential functions and pathways of key miRNA. miRNA inhibitor was transfected to detect the function of miRNA. The effect of miRNA deregulation on TSCC cell proliferation and apoptosis was investigated using MTS, Annexin V-FITC/PI double staining, and flow cytometry assays. miR-27a was a key miRNA in TSCC, which was significantly up-regulated in both Cal-27 cells and malignant tissues from the TSCC patients. In addition, functional analysis showed that miR-27a was involved in the regulation of the MAPK, ERBB, and Jak-STAT signaling pathways. Moreover, RHOA and PRKACA were potential target genes of miR-27a, suggesting them as possible mediators of the tumor-promoting effect of miR-27a. Moreover, downregulation of miR-27a inhibited cell proliferation and facilitated cell apoptosis in Cal-27 cells. Our findings strongly suggest that miR-27a could promote the tumorigenesis and development of TSCC, which makes it a potential new diagnostic marker and therapeutic target for TSCC.