Diaveridine (DVD) has widespread use in food animals due to its antibacterial synergistic effects. This study revealed the metabolism, excretion, and tissue elimination of DVD in swine, chickens, and rats following oral gavage of 10 mg/kg b.w. tritium-labeled DVD using radioactive tracing coupled with liquid chromatography–electron spray ionization–ion trap–time-of-flight–mass spectrometry (LC–ESI–IT–TOF/MS). The metabolic pathways involved demethylation, α-hydroxylation, glucuronidation, and sulfonylation and produced four metabolites in swine (M0, DVD; M1, 3’/4′-demethyl-DVD; M2, 3’/4′-demethyl-DVD-O-glucuronide; M4, 2/4-glucuronidated-DVD) and five in chickens (M0∼M2; M3, α-hydroxy-DVD; M4) and rats (M0∼M3; M5, 3’/4′-demethyl-DVD-O-sulfation). M0 was dominant in the excreta of chicken and female and male rats, while M2 was mainly excreted in swine. Among the three species studied, M0 was the most persistent in the kidneys (t 1/2 3.15–3.89 d); therefore, M0 kidney levels are residue monitoring targets. This study enabled a thorough comprehension of the metabolism and pharmacokinetic characteristics of DVD in animals.