Yeast manganese (Mn) trafficking transporter for mitochondrial Mn-containing superoxide dismutase (MnSOD), yMTM1, belongs to mitochondrial carrier family (MCF) and is crucial for yeast MnSOD (ySOD2) activation. Arabidopsis AtMTM1 and AtMTM2 are homologs of yMTM1 and share conserved MCF motif sequence. We confirmed that AtMTM1 and AtMTM2 interacted with AtMnSOD (AtMSD1) in mitochondria and recovered ySOD2 activity in yMTM1-mutant cells. The redundant AtMTM1 and AtMTM2 have different gene expression patterns in tissues and methyl viologen (MV)-induced oxidative stress and also responded to most metal stresses along with AtMSD1. Bioassay revealed the contrasting root phenotype in microRNA-mediated AtMTM1 mutant (mtm1-i) and AtMTM2-null mutant (mtm2) under MV stress, and Mn supplement complemented the root lengths in single and mtm1-i mtm2-double mutants. We found decreased MnSOD activity was accompanied by increased FeSOD activity in double mutant. Transient expression of chloroplast-destined AtMSD1 highlighted that unidentified factors participated in AtMSD1 activation. Besides, the exogenous-expressed AtMSD1 activity was decreased in double mutant, and inductively coupled plasma optical emission spectrometry results showed that AtMTM1 and AtMTM2 involved in Mn and Fe homeostasis with a reciprocal regulation. Overall, AtMTM1 and AtMTM2 are important for MnSOD metalation and ion homeostasis, and their physiological regulations may stretch across mitochondria and chloroplasts.