With the changing environmental conditions due to natural and anthropogenic activities, plants are inevitably subjected to various biotic and abiotic stresses. These create immense pressure for their survival during unfavourable conditions. Drought is a significant abiotic stress that hinders plant development and productivity. Metallothioneins (MTs) are cytoplasmic proteins that actively regulate metal homeostasis, detoxification, developmental processes, and different plant abiotic stresses. Mustard is an economically important oilseed crop confronting drought stress at different life cycle stages. This study emphasizes the role of the chickpea Metallothionein1 (MT1) gene for drought stress tolerance in mustard (Brassica juncea L.). The qRT-PCR analysis showed a higher expression level of chickpea MT1 gene up to 10 to 14 folds in T1 generation transgenic lines of mustard under drought stress as compared to wild-type control plants. Further, the various antioxidant enzymes ascorbate peroxidase (APX), catalase (CAT), glutathione S-transferase (GST), superoxide dismutase (SOD), monodehydroascorbate reductase (MDHAR) showed enhanced activities in developed over-expressing lines. The low levels of stress markers, TBARS and H2O2, were reported during the study, enabling plants to sustain under harsh conditions. Moreover, the physiological parameters water use efficiency (WUE), stomatal conductance (gs), photosynthesis rate (A), PSII (Fv/Fm), and transpiration (E) were also found improved in transgenic lines, which assisted in enduring varying stressed conditions. The study concludes that the over-expression of the chickpea MT1 gene increases the drought tolerance capacity in mustard by regulating biochemical and physiological traits. Therefore, with the aid of genetic engineering, this could be further explored to develop crop varieties with improved stress tolerance under drought conditions.