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Background: Elevated ultraviolet-B (UV-B) radiation is potentially deleterious to many organisms specifically crop plants and has become a global challenge. Rice is an exceptionally important staple food which is grown worldwide, and many efforts have been done recently to improve rice varieties against UV-B stress. This current study aims to investigate the effects of exogenous application of β-sitosterol (βSito) on growth improvement and tolerance level of rice plants against prolonged UV-B stress. The physiological and metabolic responses were evaluated in rice plants not supplemented with βSito (Nβ) and those supplemented with βSito (Sβ)., Results: The Nβ and Sβ plants were grown under non-stress (ns) and under prolonged UV-B stress (uvs) conditions and termed as Nβ ns , Sβ ns and Nβ uvs , Sβ uvs , respectively. The application of βSito contributes positively under non-stress and specifically to UV-B stress in terms of improving numerous physiological parameters associated with growth and development such as shoot and root length, RWC, whole plant biomass, chlorophyll pigments, and photosynthetic-related parameters (Pn, Gs, Tr, WUEi, Fv/Fm, and NPQ) in Sβ compared with Nβ plants. Moreover, enhanced oxidative stress tolerance of Sβ uvs vs. Nβ uvs plants under stress was attributed to low levels of ROS and substantial trigger in activities of antioxidant enzymes (SOD, POD, CAT, and APX). Metabolic analysis was performed using GC-TOFMS, which revealed higher accumulation of several key metabolites including organic acids, sugars, amino acids, and others in Sβ uvs vs. Nβ uvs plants, which were mainly reduced in Nβ plants under stress vs. non-stress conditions., Conclusion: These results provide useful data regarding the important role of βSito on growth maintenance and modulation of several metabolites associated with osmotic and redox adjustments during UV-B stress tolerance in rice plants. Importantly, βSito-regulated plasticity could further be explored specifically in relation to different environmental stresses in other economically useful crop plants.