ATP-sensitive potassium (K_ATP) channels couple cellular metabolic status to changes in membrane electrical properties. Caffeine (1,2,7-trimethylxanthine) has been shown to inhibit several ion channels; however, how caffeine regulates K_ATP channels was not well understood. By performing single-channel recordings in the cell-attached configuration, we found that bath application of caffeine significantly enhanced the currents of Kir6.2/SUR1 channels, a neuronal/pancreatic K_ATP channel isoform, expressed in transfected human embryonic kidney (HEK) 293 cells in a concentration-dependent manner. Application of non-selective and selective PDE inhibitors led to significant enhancement of Kir6.2/SUR1 channel currents. Moreover, the stimulatory action of caffeine was significantly attenuated by KT5823, a specific PKG inhibitor, and to a weaker extent, by BAPTA/AM, a membrane-permeable Ca²⁺ chelator, but not by H-89, a selective PKA inhibitor. Furthermore, the stimulatory effect was completely abrogated when KT5823 and BAPTA/AM were co-applied with caffeine. In contrast, the activity of Kir6.2/SUR1 channels was decreased rather than increased by caffeine in cell-free, inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels were suppressed regardless of patch configurations. Caffeine also enhanced the single-channel currents of recombinant Kir6.2/SUR2B channels, a nonvascular smooth-muscle K_ATP channel isoform, though the increase was smaller. Moreover, bidirectional effects of caffeine were reproduced on the K_ATP channel present in the Cambridge rat insulinoma G1 (CRI-G1) cell line. Taken together, our data suggest that caffeine exerts dual regulation on the function of K_ATP channels: an inhibitory regulation that acts directly on Kir6.2 or some closely associated regulatory protein(s), and a SUR-dependent stimulatory regulation that requires cGMP-PKG and intracellular Ca²⁺-dependent signaling.