Radiation-induced bystander effects may have important implications in radiotherapy, but it is still not well known if radiation-induced bystander effects can be triggered in hypoxic tumor cells and what are the related bystander signals. Using human hepatoma cells of HepG2, the present study found that micronuclei (MN) could be induced in the nonirradiated cells after treatment with conditioned medium (CM) harvested from irradiated cells under hypoxic conditions. Bystander effects were diminished when the irradiated cells were pretreated with sodium hydrosulfide (NaHS, an exogenous H₂S donor) (≤100 μM). However, the bystander effects were increased when the irradiated cells were pretreated with an inhibitor of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), the synthases of endogenous hydrogen sulfide (H₂S). Western blotting showed that the expressions of CSE and CBS were increased in the irradiated hypoxic cells, but were reduced in the CM treated bystander cells. The ratio of Bcl-2/Bax, a molecular marker of apoptosis, decreased with CM treatment time. However, the activity of caspase-3 increased in the hypoxic bystander cells, and this could be regulated by both NaHS and the inhibitor of endogenous H₂S. These results demonstrate that under hypoxic conditions irradiated hepatoma cells induce bystander responses by depressing the generation of endogenous H₂S and altering Bcl-2/Bax ratios as well as caspase-3 dependent damage in the bystander cells.