The widespread application of zinc oxide nanoparticles (ZnO NPs) in modern agriculture, despite their functional benefits, has raised serious concerns regarding cytotoxicity and environmental implications. The current study was designed to rigorously investigate the interactions of these nanoparticles with spinach (Spinacia oleracea L.) and to evaluate the plant’s multifaceted responses. To this end, following the synthesis and precise characteriza-tion of the nanoparticles utilizing advanced TEM, XRD, and FT-IR techniques, the effects of various concentra-tions (0, 35, 75, and 150 mg/L) on morphological indices (organ weight and length), anatomical features (cell wall thickness and vascular structure), and biochemical parameters (photosynthetic pigments, secondary metabolites, and antioxidant enzymes) were assessed. The findings revealed a dual, dose-dependent behavior of the nanopar-ticles; low concentrations stimulated growth, whereas high levels induced oxidative stress, triggering the activa-tion of plant defense mechanisms, including cell wall thickening and the enhancement of antioxidant system ac-tivity. By providing scientific evidence regarding the anatomical and biochemical adaptation mechanisms of spinach, this study offers a valuable framework for optimizing nano-fertilizer application, managing toxicity risks, and fostering the development of safe and sustainable agriculture.