Ethylene diamine tetraacetic acid (EDTA) is a chelating agent used worldwide for the phytoextraction of heavy metals as well as household and industrial purposes and persists in the cultivated land for a long time. In this study, the effect of EDTA in the presence and in absence of heavy metal (Chromium) was evaluated on non-hyper accumulating plant rice (Oryza sativa L.) on the hydroponic solution at the cellular and molecular level. EDTA application enhanced phytochelatin (PC) and metallothionein (MT) synthesis, which was assured by up regulation of phytochelatin and metallothionein gene in rice root. However, lower quantity of EDTA (25µM) along with chromium enhanced the chromium (Cr) uptake in root but inhibited its translocation to shoot pointing vacuolar sequestration of excessive chromium in root. One the other hand, in absence of chromium, H2O2 concentration is found to be increased which significantly enhanced electrolyte leakage and lipid peroxidation compared with control plants. The findings of this investigation exhibited that excessive PCs and MTs owing to EDTA enhance reactive oxygen species (ROS) generation in absence of heavy metal (Chromium).