The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1β-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic β-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1β activation of JNK in β-cells are largely unknown. In this study, we investigated whether Ca²⁺ plays a role for IL-1β-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca²⁺ channels suppressed IL-1β-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1β-stimulated activation of JNK1/2 as assessed by immunoblotting. Inhibition of IL-1β-induced in vitro kinase activity toward c-jun after collective L- and T-type Ca²⁺ channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse βTC3 cells. Ca²⁺ influx, specifically via L-type but not T-type channels, contributed to IL-1β activation of JNK. Activation of p38 and ERK in response to IL-1β was also dependent on L-type Ca²⁺ influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca²⁺ ionophore A23187, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca²⁺ ATPase, all caused an amplification of IL-1β-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca²⁺ [bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid-acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca²⁺/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1β-stimulated c-jun phosphorylation in INS-1 or βTC3 cells. Our data suggest that Ca²⁺ plays a permissive role in IL-1β activation of the JNK signaling pathway in insulin-secreting cells.