Testosterone deficiency is associated with poor prognosis among patients with chronic heart failure (HF). Physiological testosterone improves the exercise capacity of patients with HF. In this study, we evaluated whether treatment with physiological testosterone contributes to anti-fibrogenesis by modifying calcium homeostasis in cardiac fibroblasts and we studied the underlying mechanisms. Nitric oxide (NO) analyses, calcium (Ca2+) fluorescence, and Western blotting were performed in primary isolated rat cardiac fibroblasts with or without (control cells) testosterone (10, 100, 1,000 nmol/L) treatment for 48 hours. Physiological testosterone (10 nmol/L) increased NO production and phosphorylation at the inhibitory site of the inositol trisphosphate (IP3) receptor, thereby reducing Ca2+ entry, phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) expression, type I and type III pro-collagen production. Non-physiological testosterone-treated fibroblasts exhibited similar NO and collagen production capabilities as compared to control (testosterone deficient) fibroblasts. These effects were blocked by co-treatment with NO inhibitor (L-NG-nitro arginine methyl ester L-NAME, 100 μmol/L). In the presence of the IP3 receptor inhibitor (2-aminoethyl diphenylborinate 2-APB, 50 μmol/L), testosterone-deficient and physiological testosterone-treated fibroblasts exhibited similar phosphorylated CaMKII expression. When treated with 2-APB or CaMKII inhibitor (KN93, 10 μmol/L), testosterone-deficient and physiological testosterone-treated fibroblasts exhibited similar type I, and type III collagen production. In conclusion, physiological testosterone activates NO production, and attenuates the IP3 receptor/Ca2+ entry/CaMKII signaling pathway, thereby inhibiting the collagen production capability of cardiac fibroblasts.