Lactuca sativa (lettuce) in addition to being an important vegetable has been traditionally used for therapeutic purpose due to its valuable secondary metabolites. Rol genes of Agrobacterium are known to increase the synthesis of these versatile phytochemicals. This study was designed to enhance antioxidant and antidiabetic potential of lettuce by transformation using two constructs: harboring rol ABC genes and rol C gene separately. Transgene integration and expression was confirmed through PCR and semi-quantitative RT-PCR, respectively. Various in-vitro, in-vivo and cell line based assays were performed for comparison of transformed, untransformed and field grown plants of lettuce. Results showed 60% or 100% increase in total phenolic content for plants containing rol ABC genes or rol C gene, respectively compared to untransformed control plants. For total flavonoid content, 60% increase was noticed in both types of transformed plants. In contrast to control, total antioxidant capacity of plants transformed with rol ABC genes and rol C gene increased up till 110% and 70%, respectively. Total reducing power assay showed up till 125% higher activity in both rol ABC and rol C transformed plants compared to control. Results of lipid peroxidation, DPPH and DNA damage assays showed 50%, 80% and 60% increase of antioxidant activity for rol ABC and rol C plants, respectively, compared to untransformed plants. The extracts of both genes showed similar but significant enhancement behavior in hot plate analgesic and carrageenan induced hind paw edema test in rats. Up to 2 fold increase in activity was detected with 81% and 73% enhancement for the rol C and rol ABC genes respectively. These results suggest the dual inhibition nature and strong positive correlation between analgesic and anti-inflammatory activity. The viiitransformants of rol C gene exhibited prominent antidepressant activity with 190% increase as compared to untransformed plants. As for as the anticoagulant activity is concerned, an 80% increase in activity was found for the rol C extracts only. No enhancement in antidepressant and anti-coagulant activity was detected in the rol ABC gene extracts. Additionally, current findings show that seed extracts have the highest antidepressant activity. In-vitro bioactivity testing showed that rol ABC and rol C significantly improved the ability of L. sativa for its antidiabetic potential. In α-glucosidase enzyme inhibition assay, the transfomants of rol C showed 64% increase while rol ABC transfomants showed 100% increase in activity. In the comparative study of transformed and untransformed plants, the transfomants of rol C showed 84% increase while rol ABC transfomants showed 85% increase in their ability to inhibit DPP-4 enzyme. In case of GLP-1 secretion, the transfomants of rol C showed the greatest increase in GLP-1 level with 559% increase, on the other hand rol ABC transfomants showed 405% increase compared to the untransformed control plants. In the untargeted metabolomics study (LC-QTOF-MS), out of 5333 metabolites profiled and quantified, the levels of 3637 and 1792 significantly differed in rol ABC and rol C, respectively compared to untransformed plants. We identified 76 lettuce metabolites (including sesquiterpene lactones (12), non-phenolic (24), and phenolic (40) compounds) some of which changed by several folds. For example, the secondary metabolite ferulic acid methyl ester increased up to 115 fold. In conclusion, we propose that rol gene transformation significantly alters the metabolome of L. sativa and enhances its phytochemical, antioxidant, analgesic, anti-inflammatory and antidiabetic properties.