Voltage-dependent and calcium-activated K+ (MaxiK, BK) channels are widely expressed in many tissues and organs where they play various physiological roles. Here we report discovery of a functional trafficking signal in MaxiK channel accessory β4 subunit that could regulate activity of MaxiK α subunit (hSlo) on the plasma membrane. We demonstrate that β4 is mostly retained within the cell and removal or mutation of β4 trafficking signal significantly enhances its surface expression in HEK293T expression system. In hippocampal slices and cultured neurons we also observed significant β4 expressions within the neurons. Finally, we show that unlike SV1 and β1 subunits, β4 shows no dominant-negative effect on MaxiK channel α subunit. Taken together, we propose β4 subunit of MaxiK channel is mostly retained within the cells without interfering with other subunits. Removal of β4 retention signal increases its surface expression that may lead to reduction of the MaxiK channel activity and neuronal excitability. © 2014 Elsevier B.V.