Purpose: The study aimed to investigate the effects of chronic moderate exercise on regulation of intracellular calcium signaling as an important link to proliferation capacity in murine splenic T lymphocytes. Methods: Male CD1 Swiss mice were randomly assigned either to a control group (CG) or an exercise training group (EG). EG mice performed voluntary exercise for 3 months. Lymphocytes were isolated from murine spleens and intracellular calcium was determined by using Fura-2(AM) and fluorescence spectrometry. The combination of flow cytometry and carboxy-fluorescein succinimidyl ester labeling technique was used for determination of cell proliferation. The expression levels of Ca2+-regulating genes were determined by quantitative polymerase chain reaction (qPCR) analysis. Results: Basal [Ca2+](i) was significantly higher in mice from the EG compared with mice of the CG (P < 0.001, n = 6). Similarly, [Ca2+](i) transients after stimulation with phytohemagglutinin, concanavalin A, and the anti-CD3 antibody induced were significantly increased in mice from the EG (P < 0.05, n = 5). However, no differences were found after stimulation with thapsigargin (P < 0.05, n = 5). CD3+ T cells from EG showed higher mitogen-induced proliferation levels than from CG (P < 0.05/0.01, n = 5). The mRNA expression of cellular Ca2+-regulating genes, such as STIM1, Cav2.3, TRPV4, IP3R2, ORAI1, MCU, TRPM5, and TRPC1, were significantly downregulated (P < 0.05/0.01, n = 5). Conclusion: This study suggests that chronic moderate exercise improves intracellular Ca2+ signaling in murine splenic lymphocytes. The enhanced availability of the second messenger Ca2+ is followed by an improved cellular function such as cell proliferation. The downregulation of Ca2+ homeostasis-related factor expression might be considered as a self-protective mechanism against elevated intracellular Ca2+ signals.