The aim of this study was to determine whether testicular volume is correlated with clinical and biochemical markers of hypothalamic-pituitary-testicular (HPT) axis function. This was a cross-sectional substudy of a larger randomized controlled trial including obese men, body mass index (BMI) ≥30 kg m-2, with a total testosterone level <12 nmol l-1. Testicular volume was measured by orchidometer, testosterone by liquid chromatography/tandem mass spectrometry, and body composition by dual-energy X-ray absorptiometry. Men completed the Aging Males' Symptoms (AMS) score, International Index of Erectile Function-5 (IIEF-5), physical function, and handgrip dynamometer testing. Eighty-nine men participated with a median (interquartile range [IQR]) age of 53.1 (47.6, 59.2) years, BMI of 37.0 (34.6, 40.5) kg m-2, and a total testosterone of 7.0 (6.1, 7.9) nmol l-1. Median testicular volume was 18 (IQR: 10, 20) ml. Testicular volume was negatively correlated with BMI (τ =-0.1952, P = 0.010) and total fat mass (τ =-0.2115, P = 0.005) independent of age and testosterone. When BMI, testosterone, sex hormone-binding globulin (SHBG), and luteinizing hormone (LH) were present in a multivariable model, only BMI (-0.38 ml change in testicular volume per 1 kg m-2BMI; 95% CI:-0.74,-0.02; P = 0.04) and LH (-0.92 ml change in testicular volume per 1 IU l-1 LH; 95% CI:-1.75,-0.095; P = 0.03) remained independent significant predictors of testicular volume. Testicular volume was positively correlated with IIEF-5 (τ = 0.2092, P = 0.021), but not related to handgrip strength, physical function tests, or AMS. In obese men, testicular volume is inversely and independently associated with measures of adiposity, but not with most clinical or biochemical markers of HPT axis action. From a clinical perspective, this suggests that obesity might compromise the reliability of reduced testicular volume as a sign of androgen deficiency in men.