Zn is a vital micro-nutrient in living body and food industries have been using Zn as feed additive in animal diets. Previous studies presented that the performance of organic Zn differ from inorganic Zn due to higher bioavailability of organic Zn than that of inorganic Zn, but the addition of organic Zn in animal diet is costly. Therefore, to fulfil animal’s requirements, Zn is added in excess than the normal requirement. This can leads to environmental pollution when stool with excess Zn is released outside. Also the excess dietary Zn may cause other trace elements and vitamins. NPs are regarded to be highly absorbed into the gastrointestinal systems than larger particles. ZnO NPs in lesser quantity instead of bulk-sized ZnO, can be a better option. Different zinc salts affects the intestinal ion transport. There is less information about ZnO NPs effects on intestinal transport. The current study is established to study the in vitro effects of ZnO NPs on electrophysiological properties and its flux across jejunal mucosa of laying hens. Twelve White Leghorn laying hens were procured from a local commercial farm, kept in a shed and provided with feed and water ad libitum. After a week the birds were killed and jejunum was then removed, washed and conveyed in ice-cold oxygenated buffer to the laboratory within 5 minutes. Four segments were taken out of jejunum and designated with four groups (Control, ZnO 70 µM, ZnO NPs 70 µM and ZnO 35 µM). Each of the segment were mounted on separate UC. After mounting, twenty minutes were given to the tissues for equilibration under open circuit and then short-circuited by clamping the voltage at 0 mV. After getting the base line, the ZnO NPs and ZnO were added in separate chambers. The ZnO NPs or ZnO was added to the mucosal compartment of the Ussing chamber where its response was noted. Glucose was added after the incubation of approximately twenty to twenty five minutes. The electrical response was measured as the peak response obtained two minutes after the addition of glucose. The change in Isc (∆Isc) was estimated after addition of ZnO, ZnO NPs and glucose. Presentation of data was done as means ± S.E.M. The data was analyzed using One-Way Analysis of variance. The group differences were compared by the Post Hoc Tukey Test. Differences were considered significant at P < 0.05. The result showed that there was an obvious decrease in Isc of tissue treated with ZnO (Group II) but a slight increase in Isc was observed when segments treated with ZnO NPs at 70 µM and 35 µM concentration (Group III and IV). Statistically, the mean ∆Isc values of Group II was significantly different from control, Group III and IV. While Group III and IV were not significantly different from control. Also, no change existed between Group III and IV. A significant decline in glucose induced Isc was observed in tissue of Group II as compare to control, Group III and IV. There was a slight non-significant decline in glucose induced Isc when treated with ZnO NPs at 70 µM and 35 µM concentration. In conclusion, ZnO NPs are not showing any significant interference with the ionic and glucose transport and can be used as a food supplement to fulfil Zn demands. On the other hand, ZnO suppress Cl- ion secretion and glucose transport in vitro. Further confirmation of these fact needs more studies with more decreased dose of ZnO NPs.