Oxidative stress is important for cell fate. Islets of Langerhans are very sensitive to oxidative stress because of their low antioxidant levels. In this study, we investigated whether the ratio of the two main islet cell types, alpha and beta cells, and their interactions due to culturing in three-dimensional (3D) aggregates could protect against oxidative stress. Monolayer and aggregate culture were established by seeding the alphaTC1 (alpha) cells and INS1E (beta) in varying ratios and hydrogen peroxide (H2O2) was used to induce oxidative stress. Viability, oxidative stress and the level of antioxidant glutathione (GSH) were determined. Both 3D aggregation and an increasing prevalence of INS1E cells conferred greater resistance to cell death induced by oxidative stress. Increasing the prevalence of INS1E cells also decreased the number of oxidative stress positive alpha cells in monolayer culture. In 3D aggregates, the ratio of 50:50 prevented oxidative stress both in beta and alpha cells.