Mitochondrial dysfunction plays a crucial role in neurodegenerative disorders. Enhancing mitochondrial biogenesis is a promising therapeutic strategy for mitigating mitochondrial damage. Piperine, a bioactive alkaloid from black pepper, the fruit of Piper nigrum L. in the family Piperaceae, has demonstrated neuroprotective effects against mitochondrial stress. However, its effects on mitochondrial health remain unclear. This study investigated the effects of piperine on mitochondrial dynamics in SH-SY5Y neuronal cells. Our findings suggest that piperine enhances mitochondrial biogenesis by upregulating peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) mRNA and translocase of outer mitochondrial membrane 20 (TOM20) protein expression. Additionally, piperine improves Ca(2+) transport within mitochondria and boosts mitochondrial metabolic activity without significantly altering mitochondrial morphology. Furthermore, piperine prevents 6-hydroxydopamine (6-OHDA)-induced cellular stress by alleviating the activation of Homo sapiens heat shock protein family A member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3) mRNA expression and inhibiting the apoptotic Bcl-2-associated X protein (BAX) to B-cell lymphoma 2 (Bcl-2) pathway. Notably, this neuroprotective effect occurs independently of its antioxidative activity. Taken together, our results reveal a previously unexplored aspect of piperine's neuroprotective mechanism, highlighting its ability to enhance mitochondrial biogenesis and prevent mitochondrial stress in neuronal cells. Further studies, including in vivo investigations and long-term assessments, are warranted to explore the therapeutic potential for mitochondrial dysfunction in the central nervous system.