Mitochondrial apoptosis, morphology and function can be modulated by the activity of mitochondrial chaperones including mtHsp40 and mtHsp70. We found that both overexpression and depletion of mtHsp40 reversibly changed mitochondrial morphology from tubular network to fragmented puncta, in a process known as mitochondrial fragmentation, which was not coupled with apoptosis. Using domain deletion mutant constructs, we determined that mitochondrial targeting sequence (MTS) and DnaJ domain of mtHsp40 were required for mitochondrial fragmentation. Both inhibition and loss of mtHsp70 also caused mitochondrial fragmentation. Ectopic expression of mtHsp70, with the exception of substrate binding domain-deletion mutant, did not affect mitochondrial morphology. These data suggest that the stoichiometric ratio between mtHsp40 and mtHsp70 determines mitochondrial morphology independently of apoptosis. Mitochondrial fragmentation resulting from the imbalance between mtHsp40 and mtHsp70 was dependent on DRP1 level, but mitochondrial translocation of DRP1 was not detected in fragmented mitochondria. In addition, OPA1 short-isoform highly accumulated in fragmented mitochondria, suggesting that ratio between mtHsp40 and mtHsp70 is a determinant of OPA1 cleavage, which in turn determines mitochondrial morphology. Imbalance between mtHsp40 and mtHsp70 led to dramatic crista remodeling which enhances cancer cell sensitivity to drug-induced apoptosis, and lowered both ATP production and oxygen consumption rate in fragmented mitochondria, which resulted in cell-growth retardation. Collectively, we propose that perturbations of mtHsp40:mtHsp70 network might reduce their own activity, which causes OPA1 cleavage and mitochondrial fragmentation depending on DRP1 located in mitochondria, leading to mitochondrial innermembrane remodeling, indicating the apoptosis-independent coupling between mitochondrial homeostasis and mtHsp40:mtHsp70 network.