The potential toxicity and ecological risks of rare-earth nanoparticles in the environment have become a concern due to their widespread application and inevitable releases. The integration of hydroponics experiments, partial least squares structural equation modeling (PLS-SEM), and Transmission Electron Microscopy (TEM) were utilized to investigate the physiological toxicity, uptake and translocation of yttrium oxide nanoparticles (Y₂O₃ NPs) under different hydroponic treatments (1, 5, 10, 20, 50 and 100 mg·L⁻¹ of Y₂O₃ NPs, 19.2 mg·L⁻¹ Y(NO₃)₃ and control) in tomato (Lycopersicon esculentum) seedlings. The results indicated that Y₂O₃ NPs had a phytotoxic effect on tomato seedlings' germination, morphology, physiology, and oxidative stress. The Y₂O₃ NPs and soluble Y^III reduced the root elongation, bud elongation, root activity, chlorophyll, soluble protein content and superoxide dismutase and accelerated the proline and malondialdehyde in the plant with increasing concentrations. The phytotoxic effects of Y₂O₃ NPs on tomato seedlings had a higher phytotoxic effect than soluble Y^III under the all treatments. The inhibition rates of different levels of Y₂O₃ NPs in shoot and root biomass ranged from 0.2% to 6.3% and 1.0–11.3%, respectively. The bioaccumulation and translocation factors were less than 1, which suggested that Y₂O₃ NPs significantly suppressed shoot and root biomass of tomato seedlings and easily bioaccumulated in the root. The observations were consistent with the process of concentration-dependent uptake and translocation factor and confirmed by TEM. Y₂O₃ NPs penetrate the epidermis, enter the cell wall, and exist in the intercellular space and cytoplasm of mesophyll cells of tomato seedlings by endocytic pathway. Moreover, PLS-SEM revealed that the concentration of NPs significantly negatively affects the morphology and physiology, leading to the change in biomass of plants. This study demonstrated the possible pathway of Y₂O₃ NPs in uptake, phytotoxicity and translocation of Y₂O₃ NPs in tomato seedlings.