Arsenic is a ubiquitous poison that crowns the list of hazardous substances as cataloged by the Agency of Toxic Substances and Disease Registry. Arsenic occurs naturally in over 200 different mineral forms, of which approximately 60% are arsenates. Humans are exposed to pentavalent arsenicals through groundwater and ecological food chains. Absorption of sodium arsenate (As V) through the gastrointestinal tract has been reported to be 94% in humans. However, to date, no basic study has been done on the effect of As V exposure on the response of neurotransmitter pathways and enzymes related to those neurotransmission pathways in the mammalian gastrointestinal system. Intestinal motility is crucial to digestive and absorptive functions of gastrointestinal tract and as such is regulated by a myriad of excitatory and inhibitory neurotransmitters and enzymes catalyzing or synthesizing them. To bridge this knowledge gap we adopted a novel combination of crossover and placebo controlled study (1). The study was designed in two sets. In set A -in vitro organ bath experiments were conducted (following the protocols of crossover methodology) on isolated intestine of Charles foster rats (male, 35±5 day, 100 ± 17g, n=7) with a combination of different neurotransmitters and As V applied postmortem in the tissue chamber. In the first set isolated duodenal segments were first incubated with As V and the neurotransmitter in that order and also in the reversed order. The motility records were obtained by isotonic transducer IT-2245 coupled to RMS-Polyrite D software [RMS (P) Ltd, Chandigarh, India, 2010]. Our study showed that As V induced additive effect to acetylcholine and counteractive effect to nitric oxide. In set B – the test group rats (n=7) were exposed orally to LD50 dose of As V for 24 hours for a placebo controlled study, whereas the control rats were treated with arsenic free distilled water. Rats were humanely killed by cervical dislocation following the institutional guidelines foregoing anesthesia due to the nature of the experiment. In the second set we compared acetylcholinesterase activity (2) and nitric oxide synthase expression profile (3) of male Charles Foster rats (100 ± 17 g) orally exposed to LD50 24h As V to sham-treated animals. The results showed As V treatment inhibited both acetylcholinesterase and nitric oxide synthase in the duodenal smooth muscle of rat. Hence, we may conclude that As V stimulates cholinergic transmission and inhibits nitric oxide mediated neurotransmission in controlling movement of small intestine of rat efferently at the local level.