Nowadays, the exploration of zinc oxide nanoparticles (ZnO NPs) based products is booming in the various directions of bio-nanomedicine and other consumer products, but the comprehensive toxicological impact posed by ZnO NPs still remains unclear. The present study systematically investigates and correlates the toxicity evaluation of ZnO NPs in RAW 264.7 murine macrophages (in vitro) and male ICR mice (in vivo) by two different administration routes, i.e. g.i. and i.p. at different doses. The in vitro studies showed a slight rise in intracellular reactive oxygen species level (ROS), NF-κB transcription factor expression (TF) and NPs uptake at higher dose, indicating the non-toxic nature of ZnO NPs below 100 μg mL-1 doses. The in vivo results demonstrate a slight gain in body weight (BW), reduction in the organ weight, mild to severe pathological alteration in the organs depending upon NP dosage and mode of administration routes. The histopathological investigation suggests that the liver, kidney, lung, spleen, and pancreas may be the target organs for ZnO NPs according to the administration routes. Serum biochemistry assay shows an elevation in the GPT and ALP level, suggesting liver dysfunction. To our knowledge, this is the first study to report the toxic effects of ZnO NPs through i.p. administration. Further, the present work will offer a deeper understanding regarding the toxicology and in vivo behaviours of ZnO NPs in mice depending upon the various administration routes.