Background: Humans are exposed to both inorganic and organic arsenic through environmental, medicinal and occupational situations. The main source of arsenic exposure is drinking water with high levels of arsenic. Aim: This study was undertaken to investigate the gender and segmental difference in arsenate (As V) uptake by everted gut sacs of mice. Materials & methods: By using the everted gut sac technique, the serosal and mucosal uptake of Arsenate (2 mM) in both sexes of mice was studied in the intestinal segments. The Arsenate in the samples of fluid present in serosal and mucosal compartments of everted gut sacs was estimated by Hydride-Generation Atomic Absorption Spectrophotometer. Results: There was a steady increase in both serosal and mucosal uptake of Arsenate in both duodenum and ileum with a rise in the initial Arsenate concentration of the incubation medium. The mucosal uptake of Arsenate was significantly higher in duodenum than ileum (P<0.001). Both serosal and mucosal uptakes were elevated in the duodenal segment of male mice when compared to female mice except mucosal uptake in ileum. Conclusion: These results indicate that there is a gender and segmental difference in the uptake of AS (V), which can be explored pharmaceutically to reduce the arsenic toxicity in population at risk.