Mean platelet volume (MPV) can reflect platelet activity. Furthermore, high MPV is associated with thrombogenic activation and increased risk of cardiovascular disease. MPV of subjects with hypertension, hyperglycemia, or hyperlipidemia is higher compared with that of normal subjects. In contrast, the relationship between MPV and uric acid (UA) is poorly understood. The present study aims to evaluate the relationship between MPV and serum UA levels in both genders. We retrospectively studied 2104 Japanese subjects (1221 males, 883 females) undergoing general health examinations. Age, gender, body mass index (BMI), blood pressure (BP), smoking habits, alcohol intake, lipid profiles, fasting plasma glucose (FPG), high-sensitivity C-reactive protein, serum UA levels and MPV were evaluated. On the basis of the serum UA levels, the subjects were categorized into the following tertiles: 1st (Q1), 2nd (Q2), and 3rd (Q3). In males, a univariate analysis revealed that age, FPG and systolic and diastolic BP were significantly associated with MPV; in addition to these parameters, in females, UA and LDL-cholesterol correlate with MPV. Furthermore, in females, a stepwise linear regression analysis showed a significant positive correlation between UA and MPV (β = 0.059, p = 0.008). MPV in females increased gradually based on the serum UA tertile, despite adjusting for confounding variables (Q1, Q2, and Q3 values were 9.88 ± 0.70, 9.95 ± 0.73, and 10.00 ± 0.77 fL, respectively; p < 0.039). The serum UA levels were found to be a key determinant of MPV in females.