The MMACHC gene encodes for an enzyme involved in intracellular vitamin B12 metabolism, and autosomal recessive defects in MMACHC represent the most common disorder of intracellular vitamin B12 metabolism. Recent studies have identified increased levels of reactive oxygen species in cells and tissues with MMACHC dysfunction, suggesting a role for oxidative stress in disease. To investigate the link between oxidative stress and MMACHC, we exposed mice as well as human and mouse cells to hypoxia, and found significant repression of MMACHC in all investigated tissues (retina, eyecup, liver, kidney) and cell lines (HeLa, ARPE-19, human and mouse fibroblasts, 661W). Furthermore, in HeLa cells, we found transcriptional repression already at 5% oxygen, which was stable during prolonged hypoxia up to 5 days, and a return of MMACHC transcripts to normal levels only 24 h after reoxygenation. This hypoxia-induced downregulation of MMACHC was not due to altered function of the known MMACHC controlling transcription factor complex HCFC1/THAP11/ZNF143. Using in vitro RNA interference against hypoxia-induced transcription factors (HIF1A, HIF2A and REST) as well as the microRNA transcription machinery (DROSHA), we observed release of hypoxia-dependent downregulation of MMACHC expression by HIF1A and DROSHA knockdowns, whose combined effect was additive. Together, these results strongly indicate that MMACHC is a hypoxia-regulated gene whose downregulation appears to be partially mediated through both hypoxia-induced transcription factor and microRNA machinery. These findings suggest that oxidative stress could impair vitamin B12 metabolism by repression of MMACHC in healthy as well as in diseased individuals.