Chronic prednisolone treatment in renal transplant recipients (RTR) causes metabolic abnormalities, which cluster in the metabolic syndrome (MS). It also suppresses the hypothalamic–pituitaryadrenal (HPA)-axis. We investigated whether HPA-axis suppression, as measured by 24 h urinary cortisol excretion, is associated with presence of the MS and its individual components, in outpatient RTR with a functioning graft for >1 year. Urinary cortisol was measured in 24 h urine, using LC–MS/MS (LOQ 0.30 nmol/L). We included 563 RTR (age 51 ± 12 years; 54% male) at median 6.0 [IQR, 2.6–11.5] years post-transplantation. MS was present in 439/563 RTR (78%). Median 24 h urinary cortisol excretion was 2.0 [IQR, 0.9–5.1] nmol/24 h. Twenty-four hour urinary cortisol excretion was independently associated with MS presence (OR = 0.80 [95% CI, 0.66–0.98], P = 0.02). It was also independently associated with bodyweight (st.β = −0.11, P = 0.007), waist circumference (st.β = −0.10, P = 0.01), BMI (st.β = −0.14, P = 0.001), fasting triglycerides (st.β = −0.15, P = 0.001), diabetes (st.β = −0.12, P = 0.005), and number of antihypertensives used (st.β = −0.13, P = 0.003). Suppressed HPA-axis activity, as reflected by decreased 24 h urinary cortisol excretion, is associated with higher prevalence of MS and its individual components (i.e. central obesity, dyslipidemia, diabetes, hypertension) in prednisolone-treated RTR. Assessment of 24 h urinary cortisol excretion by LC–MS/MS may be a tool to monitor metabolic side-effects of prednisolone in RTR.