Large conductance Ca²⁺-activated K⁺ (BK_Ca) channels encoded by the Slo1 gene (also known as KCNMA1) are physiologically important in a wide range of cell types, and form complexes with a number of other proteins that affect their function. We performed a yeast two-hybrid screen to identify proteins that interact with BK_Ca channels using a bait derived from domains in the extreme COOH-terminus of Slo1. A protein known as membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) was identified in this screen. MAGI-1 is a scaffolding protein that allows formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is expressed in a number of tissues including podocytes of the renal glomerulus and in brain. The interaction between MAGI-1 and BK_Ca channels was confirmed by co-immunoprecipitation and GST pull-down assays in differentiated cells of a podocyte cell line and in HEK-293T cells transiently co-expressing MAGI-1a and three different COOH-terminal Slo1 variants. Co-expression of MAGI-1 with Slo1 channels in HEK-293T cells results in a significant reduction in the surface expression of Slo1, as assessed by cell-surface biotinylation assays, confocal microscopy, and whole cell recordings. Partial knock-down of endogenous MAGI-1 expression by siRNA in differentiated podocytes increased the surface expression of endogenous Slo1 as assessed by electrophysiology and cell-surface biotinylation assays, whereas over-expression of MAGI-1a reduced steady-state voltage-evoked outward current through podocyte BK_Ca channels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BK_Ca channels in kidney and possibly in other tissues.