The aim of this study was to investigate the in vitro antioxidant activity of 2,2'-dipyridyl diselenide (e) by comparing this effect with m-trifluoromethyl-diphenyl diselenide (a), p-fluor-diphenyl diselenide (b), p-chloro-diphenyl diselenide (c), and p-methoxyl-diphenyl diselenide (d) in rat liver homogenate. We also investigated if the mechanisms involved in the antioxidant property of 2,2'-dipyridyl diselenide are the same that of other diselenides. Thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC) levels were determined in rat liver homogenate, as indicators of antioxidant activity. Dehydroascorbate (DHA) reductase- and glutathione S-transferase (GST)-like activities, 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical-scavenging activities and the protection against the oxidation of Fe(2+) were determined to better understand the antioxidant property of compounds. δ-Aminolevulinic dehydratase (δ-ALA-D) activity was also carried out in rat liver homogenates, as a toxicological parameter. Compound e showed the highest potency in reducing TBARS (order of IC(50) values: e < b ≤ a < d ≤ c) and PC (order of IC(50) values: e < c ≤ b ≤ a < d) levels and lower potency in inhibiting δ-ALA-D activity than other diselenides. Compound e at all concentrations tested had no enzyme-mimetic property, but had radical-scavenging activity (≥5 μM) and protected against the oxidation of Fe(2+) (50 μM); while compounds a-d showed GST and DHA-mimetic activities and protected against the oxidation of Fe(2+), but had not radical-scavenging activities. This study indicates that (i) 2,2'-dipyridyl diselenide (e) had better in vitro antioxidant effect than other diselenides and lower inhibitory effect on δ-ALA-D activity, (ii) the presence of pyridine ring is responsible for the best antioxidant effect of this compound, and (iii) 2,2'-dipyridyl diselenide acts by different mechanisms of other diselenides.