Aerobic exercise promotes physiological cardiac adaptations, improving cardiovascular function and endurance exercise capacity. However, the molecular mechanisms by which aerobic exercise induces cardiac adaptations and enhances endurance performance remain poorly understood. Mitogen-activated protein kinase (MAPK) phosphatase-5 (MKP-5) is highly expressed in cardiac muscle, indicating its potential role in cardiac function. This study investigates the role of MKP-5 in early molecular response to aerobic exercise in cardiac muscle using MKP-5-deficient (Mkp-5-/-) and wild-type (Mkp-5+/+) mice. Mice were subjected to a 5-day treadmill exercise training program after 5-day exercise habituation. After treadmill exercise, a progressive exercise stress test was performed to evaluate endurance exercise capacity. Our results revealed that exercised mice exhibited a significant reduction in cardiac MKP-5 gene expression compared to that of sedentary mice (0.19 ± 5.89-fold; p < 0.0001). Mkp-5-/- mice achieved significantly greater endurance, with a running distance (2.81 ± 169.8-fold; p < 0.0429) longer than Mkp-5+/+ mice. Additionally, MKP-5 deficiency enhanced Akt/mTOR signaling (p-Akt/Akt: 1.29 ± 0.12-fold; p = 0.04; p-mTOR/mTOR: 1.59 ± 0.14-fold; p = 0.002) and mitochondrial biogenesis (pgc-1α: 1.56 ± 0.27-fold; p = 0.03) in cardiac muscle in response to aerobic exercise. Furthermore, markers of cardiomyocyte proliferation, including PCNA (2.24 ± 0.31-fold; p < 0.001), GATA4 (1.47 ± 0.10-fold; p < 0.001), and CITED4 (2.03 ± 0.15-fold; p < 0.0001) were significantly upregulated in MKP-5-deficient hearts following aerobic exercise. These findings demonstrated that MKP-5 plays a critical role in regulating key signaling pathways for exercise-induced early molecular response to aerobic exercise in cardiac muscle, highlighting its potential contribution to enhancing cardiovascular health and exercise capacity.