Perfluorooctanesulfonate (PFOS) as an accumulative emerging persistent organic pollutant in crops poses severe threats to human health. Lettuce varieties that accumulate a lower amount of PFOS (low-accumulating crop variety, LACV) have been identified, but the regarding mechanisms remain unsolved. Here, rhizospheric activation, uptake, translocation, and compartmentalization of PFOS in LACV were investigated in comparison with those of high-accumulating crop variety (HACV) in terms of rhizospheric forms, transporters, and subcellular distributions of PFOS. The enhanced PFOS desorption from the rhizosphere soils by dissolved organic matter from root exudates was observed with weaker effect in LACV than in HACV. PFOS root uptake was controlled by a transporter-mediated passive process in which low activities of aquaporins and rapid-type anion channels were corrected with low expression levels of PIPs (PIP1–1 and PIP2–2) and ALMTs (ALMT10 and ALMT13) genes in LACV roots. Higher PFOS proportions in root cell walls and trophoplasts caused lower root-to-shoot transport in LACV. The ability to cope with PFOS toxicity to shoot cells was poorer in LACV relative to HACV since PFOS proportions were higher in chloroplasts but lower in vacuoles. Our findings provide novel insights into PFOS accumulation in lettuce and further understanding of multiprocess mechanisms of LACV.