Large conductance calcium- and voltage- activated potassium (BK) channels play an important role in regulating diverse physiological processes ranging from neuronal excitability to the control of blood flow. Indeed, they have been implicated in a number of disorders including epilepsy, stroke, cardiovascular disease, immunity, endocrine function and hearing. Posttranslational mechanisms that control cell surface expression of BK channels are thus potential novel mechanisms for controlling BK channel function. Protein palmitoylation is emerging as an important and dynamic mechanism to control ion channel trafficking. Here, we demonstrate that BK channels are endogenously palmitoylated in HEK293 cells on multiple cysteine residues within the intracellular N-terminal S0-S1 loop. Using an imaging approach we demonstrate that this allows the S0-S1 loop to associate with the plasma membrane of cells. Functionally, site-directed mutagenesis together with membrane potential assays and patch clamp electrophysiology have revealed that palmitoylation of the S0-S1 loop does not affect, single channel conductance (slope conductance: 231 ± 4 pS, n=>3), calcium sensitivity or the voltage for half maximal activation of the channel (V0.5 MAX : 11.05 ± 9 mV, in 1 µM free calcium n=>3). Rather, that palmitoylation regulates the cell surface expression of BK channels. Fluorescent imaging using extracellular epitope-tagged channels revealed a 32 ± 5% (p<0.01, ANOVA with Tukey post hoc test; N=>3, n=>500) decrease in BK channel expression at the cell surface when palmitoylated cysteine residues were mutated to alanine. This decrease in cell surface expression was also observed in S0-S1 loop palmitoylation deficient channels in which the intracellular C-terminus is palmitoylated within an alternatively spliced insert in the BK channel (STREX decreased expression 39 ± 6%; p<0.05, ANOVA with Tukey post hoc test; N=>3, n=>500). These studies demonstrate that palmitoylation is an important determinant of BK channel cell surface expression and should allow us to address the dynamic control of BK channel palmitoylation and its physiological role.