Background: Aspartate aminotransferase (AST) is pivotal in amino acid metabolism. However, the serum activity of AST, which leaks from multiple organs, including liver and skeletal muscle, is unknown in older underweight people, who are at high risk of skeletal muscle mass loss. Therefore, we measured the serum activities of AST and alanine aminotransferase (ALT), a liver-specific transaminase, in a large, community-based cross-sectional study. Methods: Clinical parameters and lifestyles were characterized in 892,692 Japanese people with a wide range of body mass indexes (BMIs; 13–39.9 kg/m2), aged 40–74 years old, who were undergoing a medical checkup. A general linear model was used to calculate the estimated mean of serum AST (EM-AST) in each BMI category after adjustment for confounding factors, including past history of cardiovascular disease and waist circumference. Results: Severe underweight (BMI 13–14.9 kg/m2) was present in 910 subjects (0.1%). Raw serum AST showed a J-shaped relationship with BMI, which was blunted in older subjects (60–74 years), and similar, but less curved relationships were identified for raw serum ALT and gamma-glutamyl transferase. These J-shaped relationships in serum AST were not altered when subjects were classified by sex, past history of cardiovascular and cerebrovascular diseases, and habitual exercise. EM-AST showed a U-shaped relationship, with a minimum at BMI 21–22.9 kg/m2, regardless of age and waist circumference. Conclusions: High serum AST but normal serum ALT is highly prevalent in older underweight people and might reflect skeletal muscle pathology.