Di-n-butyl phthalate (DBP) is widely used in consumer products. In humans and in rats, DBP is metabolized to mono-n-butyl phthalate (MBP). MBP may also further oxidize to other metabolites of DBP. We studied the metabolic profiles of DBP in rats and humans to evaluate the similarities between the two species and between different exposure scenarios. In rats administered DBP by oral gavage, we identified MBP and three urinary oxidative metabolites of DBP: mono-3-oxo-n-butyl phthalate, mono-3-hydroxy-n-butyl phthalate (MHBP), and mono-3-carboxypropyl phthalate (MCPP). MBP, MHBP, and MCPP were also present in serum, albeit at lower levels than in urine. Statistically significant correlations (p < 0.01) existed between the concentrations of MBP and the concentrations of MHBP (Pearson correlation coefficient r = 0.82 [urine] and r = 0.96 [serum]) and MCPP (r = 0.77 [urine] and r = 0.97 [serum]). However, the concentrations of these metabolites in urine collected 6 h after dosing and in serum 24 h after dosing were not correlated, suggesting continuous metabolism of DBP and/or individual differences among rats. Serum DBP metabolite concentrations increased with the dose, whereas urinary concentrations did not. We also identified MBP, MHBP, and MCPP in the urine of four men exposed to DBP bytaking a prescription medication containing DBP, and MBP and MCPP in 94 adults with no documented exposure to DBP. In the human samples, we observed statistically significant correlations (p < 0.01) among the urinary concentrations of MBP and MCPP, although the correlation was stronger for the four exposed men (r = 0.99) than for the adults without a documented exposure to DBP (r = 0.70). Our results suggest that regardless of species and exposure scenario, MBP, the major DBP metabolite, is an optimal biomarker of exposure to DBP. In addition to MBP, MCPP and MHBP may be adequate biomarkers of exposure to DBP in occupational settings orin potential high-exposure scenarios.