Di-n-dutyl phthalate (DBP), an endocrine disruptor, is one of the most widely used phthalate esters (PAEs) in the world. It can be accumulated in seafood or agricultural products and represents a substantial risk to human health via the food chain. Thus, finding a plant which can remediate DBP but have no effects on growth is the main topic of the development of DBP phytoremediation. This study used garden lettuce (Lactuca sativa L. var. longifolia), which has a significant DBP absorption capability, as a test plant to measure phytoremediation kinetics and proteome changes after being exposed to DBP. The results show that DBP accumulated in different parts of the garden lettuce but the physiological status and morphology showed no significant changes following DBP phytoremediation. The optimal condition for the DBP phytoremediation of garden lettuce is one critical micelle concentration (CMC) of non-ionic surfactant Tween 80 and the half-life (t1/2, days), which calculated by first-order kinetics, was 2.686 days for 5 mg L−1 of DBP. This result indicated that the addition of 1 CMC of Tween 80 could enhance the efficiency of DBP phytoremediation. In addition, the results of biotoxicity showed that the median effective concentration (EC50) of DBP for Chlorella vulgaris is 4.9 mg L−1. In this case, the overall toxicity markedly decreased following phytoremediation. In the end, the result of proteome analysis showed six protein spots, revealing significant alterations. According to the information of these proteomes, DBP potentially causes osmotic and oxidative stress in garden lettuce. In addition, since DBP had no significant effects on the morphology and physiological status of garden lettuce, garden lettuce can be recommended for use in the plant anti-DBP toxicity test, and also as the candidate plant for DBP phytoremediation. We hope these findings could provide valuable information for DBP-contaminated water treatment in ecological engineering applications or constructed wetlands.