Endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response, allowing cells to recover folding capacity in the organelle. However, the overwhelming response to severe damage results in apoptotic cell death. Because of the physical proximity between ER and mitochondria, a functional interrelationship between these two organelles, including mitochondrial ATP production and apoptosis, has been suggested. The adaptive response to ER stress includes the maintenance of cellular energetics, which eventually determines cell fate. We previously demonstrated that heme oxygenase-1 (HO-1) activity protects cells against ER stress in a protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent pathway. Here, we provide evidence that PERK-mediated induction of HO-1 in murine macrophages, RAW264.7, relays ER stress to mitochondrial DNA (mtDNA) replication and function. ER stress induced by thapsigargin treatments (10-100 nM) resulted in a 2-fold increase in mtDNA contents compared with that in the untreated control. HO-1 activity on ER stress is proven to be critical for mitochondrial integrity because chemical inhibition (zinc protoporphyrin, 5-20 μM) and genetic depletion of HO-1 by small interference RNA transfection suppress the activation of transcription factors for mitochondrial biogenesis. Carbon monoxide (CO), an enzymatic by-product of HO-1 activity is responsible for the function of HO-1. Limited bioavailability of CO by hemoglobin treatment triggers cell death with a concomitant decline in ATP production. Approximately 78.1% of RAW264.7 cells were damaged in the presence of hemoglobin compared with the percentage of injured cells (26.9%) under ER stress alone. Mitochondrial generation of ATP levels significantly declined when CO availability was limited under prolonged ER stress. Taken together, these results suggest that the cellular HO-1/CO system conveys ER stress to cell survival signals from mitochondria via both the activation of transcriptional factors and functional integrity of mtDNA.