Pancreatic beta cells produce and release insulin, a vital hormone that regulates blood glucose levels, and their dysfunction contributes to the development of diabetes mellitus. Dietary zinc deficiency and exposure to the toxicant inorganic arsenic have both independently been associated with diabetes, although the effects of their combination on pancreatic beta cell health and function remain unknown. We hypothesized zinc deficiency increases the toxicity associated with arsenic exposure, causing a greater susceptibility to DNA damage and disruption of insulin production. In a cell culture model, zinc deficiency increased Ins1 gene expression and insulin production, but decreased cell proliferation. Arsenic exposure also decreased cell proliferation, and increased mRNA levels of genes involved in stress response and DNA damage. Zinc deficiency attenuated this response to arsenic and increased DNA double-strand breaks. Co-exposure did not decrease insulin levels beyond what was found with arsenic alone but did result in a further decline in cell proliferation, and increased beta cell apoptosis. These results suggest zinc deficiency and arsenic, both independently and in combination, adversely affect pancreatic beta cell health and both factors should be considered in the evaluation of health outcomes for susceptible populations.