The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca²⁺ uptake capacity. This defect is neither a consequence of global alteration in cellular Ca²⁺ homeostasis nor of the reduced driving force for Ca²⁺ internalization within mitochondria, since cytosolic Ca²⁺ transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca²⁺ uptake speed in Afg3l2^−/− cells, indicating the presence of functional Ca²⁺ uptake machinery. Our results show that the defective Ca²⁺ handling in Afg3l2^−/− cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca²⁺ elevations, hampering the proper Ca²⁺ diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2^−/− MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca²⁺ buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca²⁺ homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations.