Potassium channels have been discovered in the inner mitochondrial membrane of various cells. These channels can regulate the mitochondrial membrane potential, the matrix volume, respiration and reactive species generation. Therefore, it is believed that their activation is cytoprotective in various tissues. In our study, the single-channel activity of a large-conductance calcium-activated potassium channel (mitoBKCa) was measured by the patch-clamp technique on mitoplasts derived from mitochondria isolated from human glioma U-87 MG cells. Here, we show for the first time that mechanical stimulation of mitoBKCa channels results in an increased probability of channel opening. However, the mechanosensitivity of mitoBKCa channels was variable with some channels exhibiting no mechanosensitivity. We detected the expression of mechanosensitive BKCa-STREX exon in U-87 MG cells and hypotesize, based on previous studies demonstrating the presence of multiple BKCa splice variants that variable mechanosensitivity of mitoBKCa could be the result of the presence of diverse BKCa isoforms in mitochondria of U-87 MG cells. Our findings indicate the possible involvement of the mitoBKCa channel in mitochondria activities in which changes in membrane tension and shape play a crucial role, such as fusion/fission and cristae remodeling.