Hydrogen sulfide (H2S) has been proven to act as an antioxidant in prolonging storage of postharvest fruits and vegetables, indicating that H2S might have the potential to ameliorate chilling injury (CI). Effect of H2S fumigation released from 0.5 mM H2S donor (NaHS) on banana fruit during chilling storage followed by a ripening period was evaluated. Results demonstrated that H2S fumigation maintained higher values of lightness, peel firmness and reduced accumulation of malondialdehyde. It was also found that H2S promoted the phenylalanine ammonia lyase activity, total phenolics content and antioxidant capacity. Moreover, H2O2 and superoxide anion accumulation were reduced by H2S with up-regulated activities of guaiacol peroxidase, superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase. Further investigation on proline metabolism showed that elevated proline content which resulted from increased Δ1-pyrroline-5-carboxylate synthetase activity and decreased proline dehydrogenase activity, might be related to CI tolerance improvement. These data indicate that H2S alleviates CI of banana fruit may through the enhancement of antioxidant system and proline content.