In this study, chemical (S1) and green (S2) Copper Oxide nanoparticles (NPs) were synthesized to determine their biodistribution and toxicokinetic variances in vitro and in vivo. Both NPs significantly released Copper ions (Cu) in lymphocytes and were primarily deposited in the mononuclear phagocyte system (MPS) such as the liver and spleen in mice. In particular, S2NPs seemed to be prominently stored in the spleen, whereas the S1NPs were widely stored in more organs including the liver, heart, lungs, kidney and intestine. The circulation in the blood and fecal excretions both showed higher S2NPs contents respectively. Measurements of cell viability, Hemolysis assay, Reactive Oxygen Species (ROS) generation, biochemical estimation and apoptotic or necrotic study in lymphocytes after 24 h and measurements of body and organ weight, serum chemistry evaluation, cytokines level, protein expressions and histopathology of Balb/C mice after 15 days indicated significant toxicity difference between the S1NPs and S2NPs. Our observations proved that the NPs physiochemical properties influence toxicity and Biodistribution profiles in vitro and in vivo.