In the present study we investigated the effects of Diphenyl Diselenide (DPDS) on behavioral and biochemical parameters in Drosophila melanogaster (DM). We also tried to explore the gender differences in response to DPDS treatment. One of the salient features of the present report is that, at the highest tested concentration (3.0 mmol/kg) the survival rate in females was significantly higher (65%) as compared with male subjects (27.5%). However in alive subjects, different results pattern was observed. The locomotor activity of females significantly decreased at all tested concentrations and no statistical difference was observed in males at 0.1 and 1.0 mM. To explore the toxicological pathway of DPDS, different arrays of experiments were performed. DPDS did not cause any significant alteration in total thiol contents (NPSH) in males. While, a significant decrease was observed in females at all tested concentrations (0.1, 1.0 and 3.0 mM; p<0.005). For gender comparison purpose, its important to note that females showed 2.1 and 1.63 folds increase in SOD and CAT m-RNA expression at the highest (3.0 mM) tested concentration (as compared with male subjects). In contrast to m-RNA levels of catalase, the lowest concentration (0.1 mM) of DPDS significantly decreased (P<0.005) the enzymatic activity in females and a noticeable increase (P<0.005) at highest tested concentration (3.0 mM) was observed in male subjects. Statistically no significant change was observed in Glutathione -S- transferase (GST) activity at all tested concentrations in females. However a marked increase at 1.0 and 3.0 mM was observed in males. Furthermore, we also evaluated, HSP70 mRNA levels in females, which were increased by approximately 10.6 fold (P<0.005) as compared to male subjects. These results demonstrated significant gender-differences in terms of acute response to DPDS treatment. Mechanistically thiol oxidase (TOx) potential is one of the proposed pathways of DPDS toxicity, which can not be excluded in the present findings.