Calcineurin-mediated dephosphorylation of Drp1 regulates its translocation to mitochondria, leading to mitochondrial fragmentation. The study shows that mitochondrial depolarization, often associated with sustained cytosolic Ca²⁺ rise, activates calcineurin, which dephosphorylates Drp1, particularly at serine 637. This dephosphorylation promotes Drp1 translocation to mitochondria, facilitating fission. The process is dependent on sustained Ca²⁺ levels and calcineurin activity. Drp1, a dynamin-related GTPase, is normally localized in the cytosol but translocates to mitochondria upon dephosphorylation, where it contributes to fission. The study also demonstrates that calcineurin interacts with Drp1 and cyclophilin A, and that calcineurin inhibition blocks Drp1-dependent fission. Site-directed mutagenesis reveals that dephosphorylation of serine 637 is critical for Drp1 translocation to mitochondria. Phosphorylation at serine 637 regulates mitochondrial morphology, with dephosphorylation promoting fission. The findings highlight the role of calcineurin in mitochondrial dynamics and suggest that Ca²⁺-dependent dephosphorylation of Drp1 is a key regulatory mechanism in mitochondrial fission. The study also links mitochondrial dysfunction to Drp1 recruitment on the organelle, mediated by Ca²⁺/calcineurin-dependent dephosphorylation. This process is essential for maintaining mitochondrial morphology and function, and its disruption can lead to pathological conditions. The results provide insights into the molecular mechanisms underlying mitochondrial dynamics and their regulation by cellular signals.Calcineurin-mediated dephosphorylation of Drp1 regulates its translocation to mitochondria, leading to mitochondrial fragmentation. The study shows that mitochondrial depolarization, often associated with sustained cytosolic Ca²⁺ rise, activates calcineurin, which dephosphorylates Drp1, particularly at serine 637. This dephosphorylation promotes Drp1 translocation to mitochondria, facilitating fission. The process is dependent on sustained Ca²⁺ levels and calcineurin activity. Drp1, a dynamin-related GTPase, is normally localized in the cytosol but translocates to mitochondria upon dephosphorylation, where it contributes to fission. The study also demonstrates that calcineurin interacts with Drp1 and cyclophilin A, and that calcineurin inhibition blocks Drp1-dependent fission. Site-directed mutagenesis reveals that dephosphorylation of serine 637 is critical for Drp1 translocation to mitochondria. Phosphorylation at serine 637 regulates mitochondrial morphology, with dephosphorylation promoting fission. The findings highlight the role of calcineurin in mitochondrial dynamics and suggest that Ca²⁺-dependent dephosphorylation of Drp1 is a key regulatory mechanism in mitochondrial fission. The study also links mitochondrial dysfunction to Drp1 recruitment on the organelle, mediated by Ca²⁺/calcineurin-dependent dephosphorylation. This process is essential for maintaining mitochondrial morphology and function, and its disruption can lead to pathological conditions. The results provide insights into the molecular mechanisms underlying mitochondrial dynamics and their regulation by cellular signals.