Titanium dioxide nanoparticles (TiO2 NPs) have a wide range of applications in many fields (paint, industry, medicine, additives in food colorants, and nutritional products). Over the past decade research, TiO2 NPs have been focused on the potential toxic effects of these useful materials. In the present study, we investigated the effects of subacute exposure to TiO2 NPs on emotional behavior in adult Wistar rats, the biochemical parameters, and the histology of organs. Animals were injected intraperitoneally (ip) with TiO2 NPs (20 mg/kg body weight) every 2 days for 20 days. The elevated plus-maze test showed that subacute TiO2 NPs treatment increased significantly the anxious index (AI) compared to control group. The toxicological parameters were assessed 24 h and 14 days after the last injection of TiO2 NPs. Subacute exposure to nanoparticles increased the AST/ALT enzyme ratio and LDH activity. However, the blood cell count remained unchanged, except the platelet count increase. Histological examination showed a little inflammation overall. Moreover, our results provide strong evidence that the TiO2 NPs can induce the liver pathological changes of rats. The intraperitoneal injection of TiO2 NPs increased the accumulation of titanium in the liver, lung, and the brain. The results suggest that TiO2 NPs could alter the neurobehavioral performance of adult Wistar rats and promotes alterations in hepatic tissues.