Epidemiological studies have shown that regular consumption of fruits and vegetables is associated with reduced risk of chronic diseases. Vegetables can provide vitamins, phenolics, flavonoids, minerals and dietary fibers for optimal health benefits. However, some nutrients contained in many fruits and vegetables cannot meet of the complete nutrition need in the human body. Biotechnology has the potential to improve the nutritional value of crops. Considering the high consumption of romaine lettuce in human diet worldwide, the objective of study is to enhance the contents of vitamin C, phenolics and antioxidant activity in lettuce leaves by genetic engineering techniques. The gene expression level, vitamin C content, total phenolics, as well as total and cellular antioxidant activities were analyzed by real-time PCR, HPLC, FolinaCiocalteu, Hydro-PSC and CAA methods, respectively. The bio-fortification of genetically engineered lettuce increased vitamin C up to 48.94 A- 1.34 mg/100 g FW following the increased over-expression of At GLDH. This is almost a 3.2-fold increase as the content when compared with wild type lettuce (p < 0.05). In addition, phenolic compounds in transgenic lettuce contained 120.4 A- 1.62 mg GA equiv./100 g FW, almost double the phenolic content of the wild type. Total antioxidant activities were 735.4 A- 47.7 ?mol vitamin C equiv./100 g FW, cellular antioxidant activities were 7.33 A- 0.86 ?mol quercetin equiv./100 g FW (PBS wash) and 18.14 A- 0.68 ?mol quercetin equiv./100 g FW (No PBS wash) in transgenic lettuce, respectively, 1.5, 4 and twofold increases when compared with the wild type. This study suggests that bio-fortification by genetic engineering has great potential to improve vitamin C, phenolic contents and antioxidant activity in lettuce.