Mtm1p is essential for the posttranslational activation of manganese-containing superoxide dismutase (SOD2) in Saccharomyces cerevisiae; however, whether the same holds true for Arabidopsis thaliana is unknown. In this study, by using the yeast mtm1 mutant complementation method, we identified a putative MTM gene (AtMTM1, At4g27940) that is necessary for SOD2 activation. Further, analysis of SOD activity revealed that an SOD2 defect is rescued in the yeast mutant Y07288 harboring the AtMTM1 gene. Related mRNA-level analysis showed the AtMTM1 gene is induced by paraquat but not by hydrogen peroxide, which indicates that this gene is related to the superoxide scavenger SOD. In addition, an AtMTM1::GFP fusion construct was transiently expressed in the protoplasts, and it was localized to the mitochondria. Furthermore, sequence deletion analysis of AtMTM1 revealed that the code region (amino acid (aa) 60-198) of Mtm1p plays an important role in localization of the protein to the mitochondria. Regulation of AtMTM1 gene expression was analyzed using a fusion construct of the 1,766 bp AtMTM1 promoter and the GUS (beta-glucuronidase) reporter gene. The screen identified GUS reporter gene expression in the developing cotyledons, leaves, roots, stems, and flowers but not in the siliques. Our results suggest that AtMTM1 encodes a mitochondrial protein that may be playing an important role in activation of MnSOD1 in Arabidopsis.