Pancreatic β-cells depolarize in response to glucose and fire calcium-dependent actions potentials that trigger insulin secretion. The major current responsible for action potential repolarization in these cells is a delayed rectifier and Kv2.1 subunits are thought be a major contributor of the delayed rectifier channels. Hence, blockers of Kv2.1 channels might prolong action potentials and enhance calcium influx and insulin secretion. However, the lack of specific small molecule Kv2.1 inhibitors has hindered the testing of this mechanism. Importantly, several gating modifier peptides inhibit Kv2.1 channels in a relatively specific fashion. Hanatoxin (HaTX) and guangxitoxin-1 (GxTX-1) are examples that have been used to probe the role of Kv2.1 channels in β-cell physiology. Both HaTX and GxTX-1 strongly inhibit the Kv current of β-cells from various species, arguing that Kv2.1 subunits contribute significantly to the β-cell delayed rectifier. GxTX-1 prolongs glucose-triggered action potentials, enhances glucose-dependent intracellular calcium elevations and augments glucose-dependent insulin secretion. Taken together, these data suggest that blockers of Kv2.1 channels may be a useful approach to the design of novel therapeutic agents for the treatment of type 2 diabetes. These studies highlight the utility of gating modifier peptides in the study of physiological systems.