The B-cell lymphoma-2 (Bcl-2) family proteins are critical regulators of apoptosis and consist of both proapoptotic and antiapoptotic factors. Within this family, the myeloid cell leukemia factor 1 (Mcl-1) protein exists in two forms as the result of alternative splicing. The long variant (Mcl-1L) acts as an antiapoptotic factor, whereas the short isoform (Mcl-1S) displays proapoptotic activity. In this study, using splice-switching antisense oligonucleotides (ASOs), we increased the synthesis of Mcl-1S, which induced a concurrent reduction of Mcl-1L, resulting in increased sensitivity of cancer cells to apoptotic stimuli. The Mcl-1 ASOs also induced mitochondrial hyperpolarization and a consequent increase in mitochondrial calcium (Ca(2+)) accumulation. The high Mcl-1S/L ratio correlated with significant hyperfusion of the entire mitochondrial network, which occurred in a dynamin-related protein (Drp1)-dependent manner. Our data indicate that the balance between the long and short variants of the Mcl-1 gene represents a key aspect of the regulation of mitochondrial physiology. We propose that the Mcl-1L/S balance is a novel regulatory factor controlling the mitochondrial fusion and fission machinery.