Background Cardiac structure and function undergo progressive age-related changes influenced by sex, yet systematic characterization of these physiological variations in preclinical models remains incomplete.Methods Using echocardiography and histopathology, we examined age- and sex-dependent cardiac phenotypes in healthy adult C57BL/6J mice (n = 40), stratified into eight groups by age (8, 12, 20, and 30 weeks) and sex (male/female, n = 5 per group). Longitudinal echocardiographic assessments quantified cardiac dimensions and function, whereas histopathological analyses (hematoxylin and eosin, wheat germ agglutinin, alpha-actinin and cTnI immunofluorescence, p16INK4a and ATP2B1 immunohistochemistry, and Masson's trichrome staining) evaluated myocardial architecture, fibrosis, and molecular expression characteristics.Results Female mice exhibited significant age-dependent left ventricular dilation, increased cardiac mass, and cardiomyocyte hypertrophy, whereas males exhibited greater structural stability. Conversely, males developed pronounced interventricular septal thickening and exacerbated myocardial fibrosis at later ages (20-30 weeks). Structural protein and molecular expression remodeling may represent the underlying mechanism.Conclusion These findings underscore the critical role of age and sex in cardiac remodeling and establish a normative reference dataset for cardiac parameters in healthy adult mice. By defining robust baseline metrics, this study enhances experimental design in cardiovascular research, improving reproducibility and translational relevance of preclinical studies.