Progesterone (PROG) is a naturally occurring progestagen, which has been used to prevent preterm birth, control persistent anovulatory bleeding, and treat premenstrual syndrome in clinical practices. Studies on the metabolism of PROG have demonstrated that PROG is the precursor of other steroids such as 5 beta-pregnane-3 alpha,20 alpha-diol (PD), testosterone (T), and 17-hydroxyprogesterone. PD is the most commonly used endogenous reference compound (ERC) in the isotope ratio mass spectrometry (IRMS) analysis for doping control. It is expected that the PROG administration could affect the carbon isotope ratios (C-13/C-12, expressed as delta C-13-value) of PD and T metabolites, and lead to the false-negative or false-positive results in doping test. The influences of oral and intramuscular administration of PROG on the urinary steroid profile and carbon isotope ratios of steroids were investigated in this study. It was demonstrated that the urine concentrations and the delta C-13-values of PD were affected obviously. The depleted delta C-13-values of PD could be used to suggest PROG administration. Using PD as ERC may result in the distorted evaluation for suspicious urine sample in IRMS analysis when PROG is ingested. The 5 alpha-androst-16-en-3 alpha-ol and 11 beta-hydroxyandrosterone could be used as the alternative ERCs in case of PROG administration. The carbon isotope ratios of androsterone (An) and etiocholanolone (Etio), two T metabolites, remained unchanged throughout the excretion study, which suggested that the delta values of An and Etio could still be used as the urinary markers of T administration even when PROG was administrated.