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.