The recessive lyp allele, which harbors a defective gimap5 (GTPase of immunity-associated nucleotide binding protein 5) gene, causes spontaneous apoptosis of T lymphocytes in the biobreeding diabetes-prone strain of rats. Mechanisms underlying the pro-survival function of GIMAP5 remain unclear. In this study, we show that gimap5(lyp/lyp) T cells display diminished calcium flux in response to thapsigargin or signaling via the T cell antigen receptor. This defect is manifested in mature single positive thymocytes, where the survival defect first occurs. We also show that GIMAP5 deficiency does not affect the thapsigargin-induced calcium release from the intracellular stores but impairs subsequent calcium entry across the plasma membrane. Our findings suggest that GIMAP5 is an important regulator of calcium response in T lymphocytes and impaired calcium signaling might underlie spontaneous apoptosis of gimap5(lyp/lyp) T cells.