Perfluorooctanesulfonate (PFOS) contamination in crops threatens food safety and human health. Cultivating PFOS low-accumulation variety (LAV) crops, such as lettuce, can mitigate such risks. However, the mechanisms underlying the accumulation of PFOS in LAV remain elusive. We conducted hydroponic experiments to investigate how the root system and transpiration affect PFOS bioaccumulation in LAV in comparison with the high-accumulation variety (HAV). Additionally, two detoxification efflux carrier genes LsDTX18 and LsDTX42 were expressed in the model plant Arabidopsis thaliana to elucidate their functions in PFOS transport. The results showed that weaker root morphological characteristics restricted PFOS uptake in LAV, compared to HAV. Meanwhile, lower symplastic and apoplastic radial transfer efficiency and weaker transpiration driving force cocaused less PFOS xylem loading and root-to-shoot transport in LAV than in HAV. The young leaves with stronger transpiration accumulated more PFOS. LsDTX18 and LsDTX42, localized in the plasma membrane, regulated the xylem-to-phloem transport of PFOS in the root collar; their overexpression facilitated PFOS efflux from root xylem parenchyma cells, thus leading to less PFOS loading in xylem and subsequent root-to-shoot transport. These findings provide insights into PFOS accumulation with novel mechanisms by which LsDTXs modulate PFOS efflux in plants, laying the foundation for engineering crops with less PFOS accumulation.