The iberiotoxin-sensitive large conductance voltage- and Ca²⁺-activated potassium (BK) channels (maxi-K⁺-channels) hyperpolarize the cell membrane thus supporting Ca²⁺-entry through Ca²⁺-release activated Ca²⁺-channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK-channels, cRNA encoding Ca²⁺-insensitive BK-channels (BK^{M513I+899-903}) was injected into Xenopus oocytes with or without cRNA encoding wild type JAK2, gain-of-function ^V617FJAK2 or inactive ^K882EJAK2. K⁺-conductance was determined by dual electrode voltage clamp, and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells iberiotoxin-sensitive K⁺-current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result the K⁺-current in BK^{M513I+899-903}-expressing oocytes was significantly increased following coexpression of JAK2 or ^V617FJAK2, but not ^K882EJAK2. Coexpression of BK-channel with ^V617FJAK2 but not ^K882EJAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel and ^V617FJAK2 expressing oocytes to JAK2 inhibitor AG490 (40 µM) significantly decreased K⁺-current. Inhibition of channel insertion by brefeldin A (5 µM) decreased the K⁺-current to a similar extent in oocytes expressing BK-channel alone and in in oocytes expressing BK-channel and ^V617FJAK2. Iberiotoxin (50 nM)-sensitive K⁺-current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 µM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK-channel mRNA and protein abundance. In conclusion, JAK2 up-regulates BK-channels by increasing channel protein abundance in the cell membrane.