OBJECTIVES: This study aimed to investigate the relationship between aging and tooth loss on masseter muscle quantity and quality. MATERIALS AND METHODS: This cross-sectional study was conducted among 112 participants (in their 20s to 90s) who were independent in activities of daily living and were able to follow verbal commands. Exclusion criteria comprised participants with a lack of molar occlusal support, diseases that could affect muscle function, or temporomandibular disorder. Age, tooth loss, and weight were documented, and masseter muscle thickness (MMT) and masseter muscle echo intensity (MMEI) were measured using an ultrasonic diagnostic apparatus. A multiple regression analysis was used to determine the relationship between MMT, MMEI, aging, and tooth loss, among each sex. The significance level in the statistical analysis was p < 0.05. RESULTS: In males, aging was a significant explanatory variable for MMT (adjusted R2 = 0.27), while both aging and weight were significantly associated with MMEI (adjusted R2 = 0.54). In females, tooth loss and aging were significant explanatory variables for MMT (adjusted R2 = 0.36) and MMEI (adjusted R2 = 0.5), respectively. In both men and women, MMT and MMEI were highly correlated. CONCLUSIONS: Masseter muscles in males were more likely to be attenuated by aging than in females. The main attenuation factors were observed to differ between MMT and MMEI in women. CLINICAL RELEVANCE: In females, preservation of the natural dentition or prosthetic treatment may be effective for maintaining masseter muscle characteristics. In males, additional approaches, such as resistance exercise training, may be necessary.