OBJECTIVES: We aimed to analyze gene expression profile of tongue cancer associated with early lymph node metastasis using the cancer genome atlas (TCGA) data. STUDY DESIGN: Basic research. METHODS: A total of 515 patients with matched RNAseq data of primary tumor and clinical data from TCGA data were extracted. To compare gene expression profile between early T-stage tongue cancer with cervical lymph node metastasis and late T-stage tongue cancer without cervical metastasis, genomic data of following two groups was assessed; 1) group 1: T1/2 and N2/3 (n = 41), 2) group 2: T4 and N0 (n = 65). Using R and limma package in bioconductor program, differentially expressed genes (DEGs) were extracted. Gene ontology and pathway enrichment analysis were performed using the DAVID online tool. FFPE tissue of 285 patients were evaluated for the validation of relevant genes by imunofluorescence (IF) and immunohistochemical (IHC) stain. RESULTS: A total of 225 DEGs were found, and 50 genes were highly significant with absolute fold change over eight. Gene ontology and pathway enrichment analysis revealed that most of the upregulated genes were associated with actin cytoskeleton and included following genes: ANKRD23, NO3, PDLIM3, MUSTN1, TNNT3, MYBPC1, MB, MYH3, TTN, ACTA1, and ACTC1. When comparing tongue cancer with cN0pN0 vs. pN0pN+ using the total tongue cancer cohort of TCGA, ACTA1 was the only parameter which was associated with hidden lymph node metastasis in T1/2 (P = .019). Perineural invasion was significantly associated with high expression of ACTA1 (P < .001). IF and IHC analysis revealed that actin was overexpressed, while E-cadherin and N-cadherin were not significantly different. CONCLUSIONS: Actin associated genes, especially overexpression of ACTA1 may be associated with early regional metastasis of tongue cancer. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:813-819, 2021.