The increased application of nanomaterials in agriculture has become an environmental issue due to their toxic impact on plants. The toxicity is linked to the oxidation state of elemental species after plant uptake and recent studies have indicated copper nanoparticles (nCu) to be phytotoxic. So, this study examines the Phytotoxicity of nCu to cowpea (Vigna unguiculata) and its biotransformation using X-ray absorption near edge structure (XANES) technique. Results showed that the agglomerated nCu became phytotoxic to the root and shoot of cowpea at 250–500 mg nCu/kg treatments. Concentration-dependent effects observed for Cu accumulation in roots and shoots were at optimal at 125 mg nCu/kg. XANES analysis of root tissues revealed the biotransformation of Cu from oxidation +1 in nCu after uptake to Cu2+ species in the roots. This mechanism of biotransformation could be either a strategy for mineral storage or detoxification, which will be a subject of further study. Therefore, this study revealed the existence of biotransformation of Cu in the root tissues of cowpea.