Mitochondria play essential roles in cellular redox homeostasis and viability, and oxidative stress is known to impair mitochondrial function. In this study, we examined the effects of pyridoxine hydrochloride (vitamin B₆) on mitochondrial responses in normal human dermal fibroblasts (NHDFs) exposed to hydrogen peroxide (H₂O₂). Pre-treatment with pyridoxine hydrochloride was associated with changes in mitochondrial responses, including increased cell viability, higher PCR-based mtDNA amplifiability (an indirect indicator of mtDNA integrity), and maintenance of mitochondrial membrane potential under oxidative stress conditions. These observations indicate that pyridoxine hydrochloride influences mitochondrial responses to oxidative stress in this in vitro model, and suggest its utility for evaluating small-molecule antioxidants.