Voltage-dependent and calcium-activated K⁺ (MaxiK, BK) channels are ubiquitously expressed and have various physiological roles including regulation of neurotransmitter release and smooth muscle tone. Coexpression of the pore-forming α (hSlo) subunit of MaxiK channels with a regulatory β1 subunit (KCNMB1) produces noninactivating currents that are distinguished by high voltage/Ca²⁺ sensitivities and altered pharmacology [McManus OB, Helms LM, Pallanck L, Ganetzky B, Swanson R, Leonard RJ (1995) Functional role of the beta subunit of high conductance calcium-activated potassium channels. Neuron 14:645–650; Wallner M, Meera P, Ottolia M, Kaczorowski G, Latorre R, Garcia ML, Stefani E, Toro L (1995) Characterization of and modulation by a β-subunit of a human maxi K_Ca channel cloned from myometrium. Receptors Channels 3:185–199]. We now show that β1 can regulate hSlo traffic as well, resulting in decreased hSlo surface expression. β1 subunit expressed alone is able to reach the plasma membrane; in addition, it exhibits a distinct intracellular punctated pattern that colocalizes with an endosomal marker. Coexpressing β1 subunit with hSlo, switches hSlo’s rather diffuse intracellular expression to a punctate cytoplasmic localization that overlaps β1 expression. Furthermore, coexpressed β1 subunit reduces steady-state hSlo surface expression. Site-directed mutagenesis underscores a role of a putative endocytic signal at the β1 C-terminus in the control of hSlo surface expression. We propose that aside from its well-established role as regulator of hSlo electrical activity, β1 can regulate hSlo expression levels by means of an endocytic mechanism. This highlights a new β1 subunit feature that regulates hSlo channels by a trafficking mechanism.