Glucose is a metabolic regulator of insulin secretion from pancreatic β-cells, which is regulated by intracellular Ca2+ signaling. We and others previously demonstrated that glucose activates CD38/ADP-ribosyl cyclase (ADPR-cyclase) to produce two Ca2+ second messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Although F-actin remodeling is known to be an important step in glucose stimulated insulin secretion, the role of actin cytoskeleton in regulating Ca2+ signaling in pancreatic β-cells remain to be solved. Here, we show that actin filaments are involved in the activation of CD38/ADPR-cyclase in pancreatic β-cells. Glucose induces a sequential formation of cADPR and NAADP. Pretreatment with jasplakinolide, an actin polymerizing agent or a myosin heavy chain IIA (MHCIIA) blocker, blebbistatin, inhibited glucose-induced CD38 internalization, an essential step for cADPR formation. Blocking actin disassembly with jasplakinolide also abrogates glucose-induced cADPR and NAADP formation and sustained Ca2+ signals. These results indicate that actin filaments along with MHCIIA play an important role in CD38 internalization for the generation of Ca2+ mobilizing messengers for glucose-induced Ca2+ signaling in pancreatic β-cells.