Shiga toxin 2 (Stx2) toxoid produced by formaldehyde treatment of purified toxin was used to immunize BALB/c mice for monoclonal antibody (MAb) production. The neutralizing activities of positive clones against Stx2 were screened by in vitro cytotoxicity assay. The isotype and specificity of resultant clone was determined, and its efficacy to neutralize the activity of purified Stx2 was evaluated by in vitro and in vivo toxicity model. Lastly, its spectrum of activity against Stx2 variants was also accessed by mouse toxicity model. It was demonstrated that one of the 12 positive MAb clones against Stx2, designating S2C4 had neutralizing activity. S2C4 belongs to the immunoglobulin G1 subclass and has a κ light chain, and it reacts with the A subunit of Stx2 and does not bind to Stx2 B subunit or to Stx1. S2C4 could efficiently neutralize the cytotoxicity of Stx2 to Vero cells and mice. It also protected mice against lethal doses of Stx2 variants challenge including Stx2c and Stx2vha. S2C4 is a promising candidate molecule in preventing the progression of hemolytic-uremic syndrome (HUS) mediated mainly by Stx2 in Stx-producing Escherichia coli (STEC) infection.