Alzheimer’s disease is the most common form of dementia. This neurodegenerative disorder is characterized by the overproduction and extracellular deposition of the pathological amyloid-β (Aβ) peptides. It was shown that one of the hallmarks of Alzheimer’s disease are defects of mitochondria. We hypothesize that the mitochondrial large conductance calcium activated potassium (mitoBK(Ca)) channel could be one of the molecular targets for Aβ. It was previously found that mitoBK(Ca) is present in mitochondria of various tissues including the brain, and proved to play cytoprotective function during neuropathology. Therefore, the aim of our research was to investigate the effects of Aβ on the activity of mitoBK(Ca) channels. For our studies, mitochondria were isolated from the human astrocytoma U-87 MG cell line by the differential centrifugation. The activity of single mitoBK(Ca) channels was recorded from mitoplasts by the patch-clamp technique in the «inside-out» mode. The β1-42-amyloid (Aβ1-42) and its D-enantiomer (D-Aβ1-42) in the form of either monomers, oligomers or amyloid fibrils were added directly to the medium with the recording pipette. The probability of channel opening was determined with the Clampfit 10.3 software (Molecular Devices, USA). Detection of Aβ1-42 structural forms was performed using Thioflavin-T fluorescence assay and electron microscopy. For the first time, we showed that all three forms of Aβ1-42 – monomers, oligomers and fibrils – significantly inhibit mitoBK(Ca) channels. We used 0.1 – 0.5 µM of Aβ1-42 and described its concentration-dependent block with a partial wash-out. The strongest inhibition of mitoBK(Ca) channels was caused by oligomers, which are known to be inserted deep into lipid bilayers. Interestingly, Aβ1-42 blocked mitoBK(Ca) channels from the both sides of the membrane indicating the lack of a specific binding site in the channel molecule. Similarly, D-Aβ1-42 inhibited channel showing the lack of stereoisomer specificity. We concluded that the action of Aβ1-42 can be explained by its interaction with the mitochondrial membranes. Thus, we demonstrated that the impairment of mitoBK(Ca) channel functioning is one of the probable mechanisms of Aβ-induced cytotoxicity. These results may help to develop potential methods of the prevention and treatment of Alzheimer’s disease in the future.