The activation of mitochondrial potassium channels induces cytoprotection in various cell types. Hence, the identification of ion channels present in the inner mitochondrial membrane of keratinocytes is important in distinguishing possible protective mechanisms in these cells. In this paper, inner membrane mitochondrial ion channels of the human keratinocyte HaCaT cell line were investigated using a patch-clamp technique. We observed potassium-selective channel activity with a conductance of 83 pS at positive voltages. The I-V curve indicates that the observed channel has rectifying properties. Moreover, the channel activity was inhibited by acidic pH and 1 mM lidocaine. Using reverse transcriptase-PCR, we found an mRNA transcript for the TASK-3 (tandem pore domain acid-sensitive K channels) channel. We observed co-localization of the TASK-3 protein and a mitochondrial marker in the mitochondria of HaCaT cells. Additionally, we showed that TASK-3 knockdown HaCaT cells markedly decreased viability after UVB radiation exposure compared with control cells. In summary, the single-channel activity and properties of a mitochondrial potassium channel in a keratinocyte HaCaT cell line have been described.