The objective of this study was to comparatively evaluate the anti-inflammatory effects of intravenous administration of insulin (in different doses) and flunixin on physiological variables, acute-phase responses, and cardiovascular biomarkers during inflammatory phase which was induced by Escherichia coli lipopolysaccharide (LPS) serotype O55:B5 in miniature donkeys. A total of twenty-four clinically healthy male adult miniature donkeys aged 5 ± 1 year and weighing 120 ± 10 kg were studied. The animals were assigned randomly to four experimental groups (three treatment and one positive control groups). Six hours after induction of inflammatory phase (by 20 ng kg-1 LPS), donkeys in groups Insln 1.5, Insln 3, and Flnx received insulin at 1.5 IU kg-1, insulin at 3 IU kg-1, and flunixin at 2.2 mg kg-1, respectively. Animals in the positive control group were assigned to receive LPS without any anti-inflammatory drugs. We have shown that serum concentrations of cardiovascular, acute-phase proteins and cytokines were increased during inflammatory phase in miniature donkeys. Our results revealed that insulin at 3 IU kg-1 as well as flunixin at 2.2 mg kg-1 can improve almost all of the physiological variables and hematobiochemical variables (including serum concentrations of tumor necrosis factor-alpha, interferon-gamma, haptoglobin, serum amyloid A, cardiac troponin I, hemocysteine, white blood cell, and packed cell volume) after 24 hours. Unlike insulin at 1.5 IU kg-1, insulin at 3 IU kg-1 may be considered useful for inflammatory conditions in miniature donkeys.