The sulfite reductase gene in Medicago sativa L. (MsSiR) encodes sulfite reductase (SiR) and catalyses the conversion of sulfite to sulfate in the sulfite assimilation pathway. In this study, we investigated the role of MsSiR in alfalfa by generating transgenic alfalfa that ectopically expressed MsSiR under the control of the CaMV35S promoter. The differences in alkali tolerance between the MsSiR-overexpressing and wild-type (WT) plants were analyzed, and the MsSiR-overexpressing plants exhibited an improved phenotype under alkali stress. Compared to WT plants, these plants demonstrated improved antioxidant activity as well as decreased HO and O contents and increased glutathione reduced (GSH), Cysteine (Cys) and glutathione oxidized (GSSG) contents. MsSiR-overexpressing plants also exhibited high levels of adenosyl phosphosulfate reductases (APR), sulfite oxidase (SO) and MsSiR expression under alkali stress. It was speculated that MsSiR is involved in sulfur metabolism pathways, including the stabilization of sulfate and sulfite levels and the synthesis of GSH. These two processes achieve alkali tolerance by positively regulating the detoxification and antioxidant activities of alfalfa.