High blood glucose triggers the release of insulin from pancreatic beta cells, but if chronic, causes cellular stress, partly due to impaired Ca2+ homeostasis. Ca2+ influx is controlled by voltage-gated calcium channels (Ca-V) and high density of Ca-V in the plasma membrane could lead to Ca2+ overload. Trafficking of the pore-forming Ca-V alpha(1) subunit to the plasma membrane is regulated by auxiliary subunits, such as the Ca-V beta(2a) subunit. This study investigates, using Ca2+ imaging and immunohistochemistry, the role of palmitoylation of Ca-V beta(2a) in maintaining Ca2+ homeostasis and beta cell function. RNA sequencing data showed that gene expression of human CACNB2, in particular CACNB2A (Ca-V beta(2a)), is highest in islets when compared to other tissues. Since Ca-V beta(2a) can be regulated through palmitoylation of its two cysteines, Ca-V beta(2a) and its mutant form were overexpressed in pancreatic beta cells. Palmitoylated Ca-V beta(2a) tethered to the plasma membrane and colocalized with Ca(V)1.2 while the mutant form remained in the cytosol. Interestingly, Ca-V beta(2a) overexpression raised basal intracellular Ca2+ and increased beta cell apoptosis. Our study shows that palmitoylation of Ca-V beta(2a) is necessary for Ca-V alpha(1) trafficking to the plasma membrane. However, excessive number of palmitoylated Ca-V beta(2a) leads to Ca2+ overload and beta cell death. (C) 2017 Elsevier Inc. All rights reserved.