Recurrence and metastasis of hepatocellular carcinoma (HCC) after radiotherapy are frequently observed in clinical practice. To date, the involved mechanism, endogenous hydrogen sulfide (H₂S), has not been well understood and warrants investigation. Here we demonstrated that both single-dose and fractionated irradiation enhanced metastasis of HCC cells both in vitro and in vivo at 20–60 days postirradiation. In particular, a gain in epithelial-mesenchymal transition (EMT) and mesenchymal features was observed. Further experiments revealed that endogenous H₂S signaling was constitutively activated after irradiation. Knockdown of cystathionine-γ-lyase (CSE) or cystathionine-β-synthase (CBS), two main H₂S-producing proteins, significantly diminished the increased expressions of EMT-related proteins induced by radiation through the p38MAPK pathway, leading to impaired invasion and metastasis of the residual HepG2 cells and their xenograft tumors. Moreover, blocking of the H₂S pathway increased the radiosensitivity of the HepG2 xenograft tumor. Collectively, our results strongly suggest that endogenous H₂S/CSE contributes to the long-term cell invasion and tumor metastasis induced by fractionated exposures and therefore, could become an attractive therapeutic target of HCC to eliminate radiotherapy-induced adverse effects.