Low serum bilirubin levels have been associated with increased risk of cardiovascular disease (CVD) and metabolic syndrome. Testosterone deficiency could also contribute to increased risk of CVD and metabolic syndrome. Therefore, this study aimed to examine the relationship between serum bilirubin level and testosterone deficiency in 1284 Korean men aged 45 to 70 years. Serum bilirubin level was categorized into quartiles: Q1 ≤ 0.7, Q2 0.8-0.9, Q3 1.0-1.1, and Q4 ≥ 1.2 mg/dL. Testosterone deficiency was defined as level less than 8.0 nmol/L, as suggested by the position statement of International Society of Andrology. The overall prevalence of testosterone deficiency was 5.8% and significantly decreased with the quartiles from Q1 to Q4. Compared with the referent fourth quartile (serum bilirubin ≥ 1.2 mg/dL), the ORs (95% CIs) for testosterone deficiency was 2.29 (1.04-4.94) for the first quartile after adjusting for age, fasting glucose, triglyceride, HDL-cholesterol, leukocyte count, hemoglobin, smoking status, and alcohol intake. We found inversely graded associations of serum bilirubin level with testosterone deficiency. These findings suggest that low bilirubin level may be interpreted as a state of testosterone deficiency in middle-aged and older men.