Redroot pigweed (Amaranthus retroflexus L.) is a dominant weed in soybean [Glycine max (L.) Merr.] fields in Heilongjiang Province, China. High selective pressure caused by the extensive application of the protoporphyrinogen oxidase (PPO)-inhibiting herbicide fomesafen has caused A. retroflexus to evolve resistance to this herbicide. Two susceptible and two resistant populations (S1, S2, R1, and R2) were selected in this study to illustrate the target-site resistance mechanism in resistant A. retroflexus. Whole-plant bioassays indicated that R1 and R2 had evolved high-level resistance to fomesafen, with resistance factors of 27.0 to 27.9. Sequence alignment of the PPO gene showed an Arg-128-Gly substitution in PPX2. The basal expression differences of PPX1 and PPX2 between the S1 and R1 plants were essentially nonsignificant, whereas the basal expression of PPX2 in R2 plants was slightly lower than in S1 plants. Compared with the PPX1 gene, the PPX2 gene maintained higher expression in the resistant plants after treatment with fomesafen. An enzyme-linked immunosorbent assay showed a similar basal PPO content between the susceptible and resistant plants without treatment. After fomesafen treatment, the PPO content decreased sharply in the susceptible plants compared with the resistant plants. Furthermore, after 24 h of treatment, the resistant plants showed increased PPO content, whereas the susceptible plants had died. The PPO2 mutation resulted in high extractable PPO activity and low sensitivity to fomesafen along with changes in PPO enzyme kinetics. Although the mutant PPO2 exhibited increased K_m values in the resistant plants, the V_max values in these plants were also increased. Changes in the properties of the PPO enzyme due to an Arg-128-Gly substitution in PPX2, including changes in enzyme sensitivity and enzyme kinetics, are the target-site mechanism of resistance in A. retroflexus.