Diosmin (diosmetin-7-O-rutinoside) and its aglycone diosmetin, natural bioflavonoids distributing in a variety of citrus fruits and Chinese herbal medicines, possessed positive effects against hepatic, renal, lung, gastric, cerebral and cardiac injury. However, the in vivo metabolic profiles of diosmin and diosmetin in urine, plasma and feces still remain ambiguous. In this study, metabolites of diosmin and diosmetin were identified using an UHPLC-LTQ-Orbitrap MSn strategy coupled with multiple metabolite templates, extracted ion chromatograms (EICs) and diagnostic product ions (DPIs). As a result, 46 diosmetin metabolites and 64 diosmin metabolites were respectively identified in rat biological samples. Methylation, demethylation, hydroxylation, glycosylation, glucuronidation, diglucuronidation and sulfation were common metabolic pathways of diosmetin and diosmin, while demethoxylation, decarbonylation, dihydroxylation and dehydroxylation were particular metabolic pathways of diosmin comparing with that of diosmetin. Diosmetin was not detected in all the biological samples, suggesting that it was quickly transformed into other metabolites in vivo. Diosmin and diosmetin-7-O-glucoside identified in urine and feces as well as their subsequent metabolites accounted for a substantial part of all the diosmin metabolic products. Metabolic profiles of diosmetin and diosmin indicated that they were primarily excreted through the urine route possibly originating from the dominant role of their phase II metabolism in vivo. Our results have provided a better understanding of the similarities and differences in pharmacodynamics and pharmacokinetics of diosmetin and diosmin in the future.