BK channels control diverse physiological functions including neuronal excitability and thus mechanisms that control cell surface expression of these channels are important determinates of function in health and disease. We have recently identified that the cell surface expression of BK pore-forming α subunit is potently regulated by s-acylation, the only reversible lipid modification of proteins. In many neurons, α subunits assemble with accessory β-subunits to modify channel properties, however, whether these β-subunits may also be regulated by s-acylation to control trafficking or function is not known. Using the CSS-Palm 2.0 algorithm, we predicted the neuronal β4-subunit is a palmitoylated membrane protein and confirmed this biochemically using 3H-palmitate incorporation and acyl-RAC of β4-subunits expressed in HEK293 cells and neuronal N2a cells. Using quantitative immunoflureoscent imaging assays, wild type β4 expressed in HEK293 or N2a cells alone displayed robust cell-surface expression. However, mutation of a single predicted palmitoylated cysteine to alanine (β4-mut) resulted in enhanced trapping in the endoplasmic reticulum (ER colocalisation using Pearson’s R value was increased from 0.36 to 0.83) associated with a significant decrease (> 50%) in cell surface expression without significant effects on total β4-subunit expression. To investigate whether palmitoylation of the β4-subunit controlled trafficking of the α subunit, we co-expressed β4 with an N-terminal Flag tagged α subunit construct (Flag-ZERO) in both HEK293 and N2a cells, and then monitored α subunit surface expression and ER co-localisation. Cell surface expression of Flag-ZERO was significantly enhanced in both HEK293 (by 50 ± 5.7%) and N2a (by 90 ± 8.1%) cells upon co-expression of Flag-ZERO with β4. This was associated with a decreased ER co-localization of Flag-ZERO upon β4 expression (R value shifted from 0.80 to 0.48 in both HEK293 and N2a cells). However, this enhanced cell surface expression of Flag-ZERO promoted by β4 was dependent upon the palmitoylation of β4. Co-expression of Flag-ZERO with β4 -mut resulted in no significant enhancement of Flag-ZERO surface expression in either HEK293 or N2a cells. Therefore, the accessory β4-subunit is a palmitoylated membrane protein that regulates the trafficking of BK channel to the cell surface in a palmitoylation dependent mechanism. Dynamic regulation of accessory β4-subunit palmitoylation may have important functional consequences for BK channel function in the control of neuronal excitability.β4-subunit palmitoylation may have important functional consequences for BK channel function in the control of neuronal excitability. β4-subunit palmitoylation may have important functional consequences for BK channel function in the control of neuronal excitability.