The migration of macrophages and lymphocytes that produce cytokines such as tumor necrosis factor-α (TNF-α) causes β-cell death, leading to type 1 diabetes. Similarly, in type 2 diabetes, the adipocyte-derived cytokines including TNF-α are elevated in the circulation, causing inflammation and insulin resistance. Thus, the studies described in this article using TNF-α are relevant to furthering our understanding of the pathogenesis of diabetes mellitus. We used RINr1046-38 (RIN) insulin-producing β-cells, which constitutively express calbindin-D28k, to characterize the effect of TNF-α on apoptosis, replication, insulin release, and gene and protein expression. Western blots of TNF-α-treated RIN cells revealed a decrease in calbindin-D28k. By ELISA, TNF-α-treated β-cells had 47% less calbindin-D28k than controls. In association with the decline in calbindin-D28k, TNF-α treatment of RIN cells led to a 73% greater increase in changes in intracellular calcium concentration (Δ[Ca2+]i) in TNF-α-treated cells as compared to that in control RIN cells upon treatment with 50 mM KCl; caused a greater increase in the [Ca2+]i following the addition of 5.5 μM ionomycin; increased by more than threefold the apoptotic rate, expressed as the percentage of TUNEL-positive nuclei to total nuclei; decreased the rate of cell replication by 36%; and increased and decreased selectively the expression of specific genes as determined by microarray analysis. The subcellular localizations of Bcl-2, an antiapoptotic protein, and Bax, a proapoptotic protein, within RIN cells were altered with TNF-α treatment such that the two were colocalized with mitochondria in the perinuclear region. We conclude that the proapoptotic action of TNF-α on β-cells is manifested via decreased expression of calbindin-D28k and is mediated at least in part by [Ca2+]i. © 2005 Wiley-Liss, Inc.