Objective: To identify the changes of gene expression profiles and metastasis ability of rat hepatoma cell line McA-RH7777 after sublethal irradiation, and to explore the underlying molecular mechanism. Methods: Rat hepatoma McA-RH7777 cells were received single exposure of 6 Gy X-ray. Then, the remaining McA-RH7777 cells were continuously passaged and named as McARH7777- 6Gy. The gene expression profiles of McA-RH7777 and McA-RH7777-6Gy cells were detected by gene microarray and compared. Subsequent bioinformatic analysis of the genes with significant changes in expression levels were performed by DAVID software (including GO-analysis and KEGG-analysis). The mRNA expression levels of tissue inhibitor of metalloproteinase 2 (TIMP2), SMAD family member 2 (SMAD2) and MET proto-oncogene in McA-RH7777 and McA-RH7777-6Gy cells were detected by real-time fluorescent quantitative PCR to verify the difference in gene expression profiles of the two cells. The migration and invasion abilities of McA-RH7777 and McA-RH7777-6Gy cells were detected by scratch wound healing assay and Transwell chamber assay, respectively. Results: The gene microarray showed that the expressions of a series of tumor-related genes were changed in McA-RH7777-6Gy cells as compared with wild-type McA-RH7777 cells. Real-time fluorescent quantitative PCR showed that the relative expression levels of TIMP 2, SMAD 2 and MET in McA-RH7777-6Gy cells were raised by 13.000, 14.516 and 6.384 times as compared with McA-RH7777 cells, respectively. GO analysis showed that the remarkably changed genes mainly located in tumor metastasis-associated biological processes, such as Ras protein signal transduction, cell cycle arrest, cell migration, cell adhesion, negative regulation of apoptotic process, positive regulation of cell proliferation, positive regulation of epithelial to mesenchymal transition and positive regulation of MAP kinase activity. KEGG analysis showed these genes mainly involved in proteoglycans signaling pathway, phosphoinositide-3 kinase (PI3K)-protein kinase B (PKB, Akt) signaling pathway, viral carcinogenesis pathway, FoxO signaling pathway, Rap1 signaling pathway, Hippo signaling pathway, Ras signaling pathway, etc. The scratch healing and Transwell chamber tests showed that the migration and invasion abilities of McA-RH7777-6Gy cells were significantly enhanced as compared with McA-RH7777 cells (both P < 0.001). Conclusion: After sublethal radiation, the migration and invasion abilities of hepatoma cells are significantly enhanced, which may be related to the changes of gene expressions in metastasis-associated pathways.