Chilling injury (CI) in postharvest crops associated with an imbalance between reactive oxygen species (ROS) and antioxidant defense systems. This study investigated the role of exogenous trehalose in mitigating CI by modulating ROS and antioxidant activities in ‘Kim Ju’ guava during low temperature storage (8 °C for 14 days) followed by shelf storage (25 °C for 4 days). The findings revealed a progressive rise in CI index and ROS level, encompassing superoxide radical and hydrogen peroxide (H2O2), in untreated fruits throughout the storage duration. Concurrently, the total antioxidant capacity, activities of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX), along with the redox status of non-enzymatic antioxidants, including the ratios of ascorbic acid/dehydroascorbate (ASA/DHA) and reduced glutathione/oxidized glutathione (GSH/GSSG), exhibited a declining trend during storage, except activity of peroxidase (POX). Trehalose treatment (immersion in 200 mM for 30 min) effectively reduced CI throughout the storage period. This reduction was associated with the upregulation of total antioxidant capacity, SOD, CAT, APX and GPX activities, along with increased total phenolic content and elevated ratios of ASA/DHA and GSH/GSSG, compared to untreated fruits. Additionally, POX activity and H2O2 level were significantly lower in trehalose-treated fruits. The results of this study indicate that low temperature stress causes an increase in ROS level and a decrease in enzymatic and non-enzymatic Chiang Mai Journal of Science 2 http://epg.science.cmu.ac.th/ejournal/ Chiang Mai Journal of Science Chiang Mai J. Sci., 2025; 52(2): e2025015 DOI 10.12982/CMJS.2025.015 antioxidant activities resulting in oxidative stress and leading to CI development in control fruit. On the other hand, application of trehalose can upregulate total antioxidant capacity including enzymatic and non-enzymatic antioxidant activities to overcome ROS leading to mitigating CI guava fruit.