Fibroblast growth factor (FGF) 21 has various functions, including glucose and lipid metabolism, yet the biology of FGF21 remains unclear. This study aimed to investigate specific conditions that might affect the functions of FGF21. Subjects included 398 healthy men who underwent health examinations to obtain information on physical and biochemical parameters and lifestyle behaviors. Associations of serum FGF21 levels with each parameter were assessed in the study. FGF21 levels correlated with age, body mass index, waist circumference (WC), systolic blood pressure (SBP), diastolic blood pressure, aspartate aminotransferase, alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (γ-GTP), uric acid, total cholesterol (TC), triglycerides, high-density lipoprotein cholesterol, fasting plasma glucose, and HbA1c. Moreover, FGF21 levels were significantly associated with lifestyle behaviors, including smoking status and breakfast and alcohol consumption frequency. Multivariable regression analysis showed that age, ALT, γ-GTP, smoking status, and breakfast and alcohol consumption frequency were independent variables for FGF21 levels. Assessment among the non-obese and obese groups showed that FGF21 levels correlated with WC, SBP, and TC only in the non-obese group. Thus, serum FGF21 levels were affected by several factors, including lifestyle behaviors, age, and liver function. To assess the functions of FGF21 in individuals, considering these factors would be essential. (199 words)