Myc is a ubiquitous mediator of cell proliferation that transactivates the expression of various genes through E-box sites. Here we report a novel gene, mimitin (Myc-induced mitochondrial protein), that encodes a mitochondrial protein with a molecular mass of 20 kDa. We demonstrated that the transcription of mimitin is directly stimulated by c-Myc. To investigate the role of Mimitin, its expression was suppressed by the RNA interference (RNAi) technique. Whereas specific inhibition of mimitin expression did not affect cell proliferation in human cervical carcinoma, colon adenocarcinoma, and hepatocarcinoma cell lines, it did suppress cell proliferation in human glioblastoma, esophageal squamous cell carcinoma (ESCC), and embryonic lung fibroblastic cells, with the greatest suppression efficiency in ESCC cells. To investigate whether mimitin is related to tumorigenesis in ESCC in vivo, the expression of Mimitin protein in ESCC tissues was studied. Mimitin was highly expressed in 80% (28 of 35) of ESCC tumors, suggesting that high expression of Mimitin is a characteristic feature of ESCC. The expression level of Mimitin was found to be correlated with that of c-Myc and cell proliferation, but not with the histopathological grade, stage of cancer, or age of patients. Taken together, these results suggest that the novel gene mimitin is a direct transcriptional target of c-Myc, and is involved in Myc-dependent cell proliferation at least in ESCC cells.