S-acylation, the only reversible post-translational lipid modification of proteins, is emerging as a major mechanism to control ion channel function and physiology. The most common form of S-acylation, palmitoylation, results from the addition of the C16 lipid palmitate via a labile thioester bond to reactive cysteines in proteins. However, as for most other palmitoylated proteins, the enzymes controlling palmitoylation of ion channels are largely unknown. In this study we have developed an siRNA based screen and enzyme overexpression strategy in HEK293 cells to define the candidate acyl transferase (zDHHC) and acyl thioesterase (APT) enzymes that control palmitoylation of large conductance voltage-and calcium- activated potassium (BK) channels. Palmitoylation of a cluster of cysteine residues (C53:54:56) in the intracellular N-terminal S0-S1 loop of the pore-forming α-subunit was mediated by only two of the 23 zDHHCs: zDHHC 22 and 23. siRNA -mediated knockdown of zDHHC22 or 23 reduced 3H-palmitate incorporation into BK channel α-subunits by 67 ± 4% and 76 ± 8% of control respectively. These enzymes are distinct from the zDHHCs that control palmitoylation of an alternatively spliced insert (STREX) in the C-terminus of the BK channel α-subunit. In contrast, the S0-S1 site is depalmitoylated by the cytosolic APT1 and APT1-like, but not APT2 or lysozomal PPT1, thioesterases. Overexpression of APT1 or APT1-like enzymes reduced channel palmitoylation by 68 ± 2 % and 52 ± 7% of control respectively whereas the corresponding catalytically inactive mutants were without effect. Palmitoylation of the S0-S1 loop was required for efficient cell surface trafficking of the BK channel α-subunit. siRNA knockdown of zDHHC22 or 23, or overexpression of catalytically active APT1 or APT1-like thioesterases, reduced plasma membrane surface expression of the BK channel α-subunit by > 50% compared to control. This reduction in surface expression was similar to that observed with the palmitoylation deficient BK channel α-subunit mutant C53:54:56A and channel depalmitoylation resulted in retardation of the α-subunit in the trans-golgi network (TGN). This study has defined the repertoire of palmitoylating and depalmitoylating enzymes that control palmitoylation of the BK channel. Our studies show that both palmitoyl acyltransferases and acyl thioesterases display discrete substrate specificity for BK channels. Furthermore, as depalmitoylated BK channels are retarded in the TGN, reversible protein palmitoylation provides a critical check-point to regulate exit from the TGN and thus control BK channel cell surface expression.