Mechanosensitive large-conductance Ca2+-activated K+channels encoded by the Slo1 gene (BKCachannels) are expressed in podocytes. Here we show that BKCachannels reciprocally coimmunoprecipitate with synaptopodin (Synpo) in mouse glomeruli, in mouse podocytes, and in a heterologous expression system (HEK293T cells) in which these proteins are transiently expressed. Synpo and Slo1 colocalize along the surface of the glomerular basement membrane in mouse glomeruli. Synpo interacts with BKCachannels at COOH-terminal domains that overlap with an actin-binding domain on the channel molecule that is necessary for trafficking of BKCachannels to the cell surface. Moreover, addition of exogenous β-actin to mouse podocyte lysates reduces BKCa-Synpo interactions. Coexpression of Synpo increases steady-state surface expression of BKCachannels in HEK293T cells. However, Synpo does not affect the stability of cell surface BKCachannels, suggesting a primary effect on the rate of forward trafficking, and Synpo coexpression does not affect BKCagating. Conversely, stable knockdown of Synpo expression in mouse podocyte cell lines reduces steady-state surface expression of BKCachannels but does not affect total expression of BKCachannels or their gating. The effects of Synpo on surface expression of BKCaare blocked by inhibition of Rho signaling in HEK293T cells and in podocytes. Functional cell surface BKCachannels in podocytes are also reduced by sustained (2 h) but not acute (15 min) depolymerization of actin with cytochalasin D. Synpo may regulate BKCachannels through its effects on actin dynamics and by modulating interactions between BKCachannels and regulatory proteins of the podocyte slit diaphragm.