The transfer of nanoparticles (NPs) into the food chain through edible plants is of great concern. Cucumis sativus L. is a freshly consumed garden vegetable that could be in contact with NPs through biosolids and direct agrichemical application. In this research, cucumber plants were cultivated for 150 days in sandy loam soil treated with 0 to 750 mg TiO2 NPs kg(-1). Fruits were analyzed using synchrotron μ-XRF and μ-XANES, ICP-OES, and biochemical assays. Results showed that catalase in leaves increased (U mg(-1) protein) from 58.8 in control to 78.8 in 750 mg kg(-1) treatment; while ascorbate peroxidase decreased from 21.9 to 14.1 in 500 mg kg(-1) treatment. Moreover, total chlorophyll content in leaves increased in the 750 mg kg(-1) treatment. Compared to control, FTIR spectra of fruit from TiO2 NP treated plants showed significant differences (p ≤ 0.05) in band areas of amide, lignin, and carbohydrates, suggesting macromolecule modification of cucumber fruit. In addition, compared with control, plants treated with 500 mg kg(-1) had 35% more potassium and 34% more phosphorus. For the first time, μ-XRF and μ-XANES showed root-to-fruit translocation of TiO2 in cucumber without biotransformation. This suggests TiO2 could be introduced into the food chain with unknown consequences.