Purpose: Ultrasonography is useful for evaluating anatomical variations of the median nerve and aids in diagnosing carpal tunnel syndrome by assessing the cross-sectional area (CSA) at the carpal tunnel inlet. The purposes of this study were to investigate the prevalence of the bifid median nerve and to establish reference values for the CSA at the carpal tunnel inlet in healthy participants. Methods: A total of 349 Japanese participants were included (121 men and 228 women, mean age 66.3 years). Study participants underwent ultrasonography to assess the CSA of the median nerve at the carpal tunnel inlet of the bilateral wrist. First, we investigated the prevalence of the bifid median nerve in 349 participants. Then, we assessed the relation between the CSA and clinical factors (age, height, weight, body mass index, fat mass, fat-free mass, bilateral grip strength, and key pinch strength) in healthy participants. Results: Among the 349 participants, 59 (16.9%) had a bifid median nerve. Mean CSA in healthy participants (no bifid median nerve) was 9.7 ± 2.9 mm2 in women and 9.0 ± 1.8 mm2 in men (both: 9.4 ± 2.6 mm2). In women, mean CSA in subjects in their sixties was significantly higher than in younger subjects, and we found a significant positive correlation between the CSA and the fat-free mass. Conclusions: In this observational study of healthy Japanese, the prevalence of the bifid median nerve was 16.9%; the mean CSA was 9.7 ± 2.9 mm2 in women and 9.0 ± 1.8 mm2 in men. Older women had a larger CSA than did younger women. Clinical relevance: This study describes the prevalence of the bifid median nerve and normative values for the CSA of the median nerve precisely diagnose carpal tunnel syndrome to more by ultrasonography. Key words: bifid median nerve, cross-sectional area, median nerve, reference values, ultrasonography