Objective To explore the feasibility of high performance liquid chromatography (HPLC) in determination of paraquat concentration in rabbit serum, and to observe the characteristics of the concentration changes after intragastric administration of paraquat of different dosages in rabbits. Methods Twelve New Zealand rabbits were randomly divided into two groups, with 6 rabbits in each group. The rabbits were orally given different dosages of paraquat to induce acute toxicosis, their serum was collected after administration for 1, 2, 5, 12, 24, 48 and 72 h respectively and each serum sample was deproteinized with acetonitrile. The concentration of paraquat in serum was determinated by HPLC. The mobile phase was 0.1 mol/L phosphate buffer (12.5 mmol/L sodium heptanesulfonate)-acetonitrile (90:10), pH was adjusted to 2.8, the flow rate was 1.0 ml/min, the detector wavelength was 258 nm, column temperature was at 30℃ . Results Excellent linear relationship was obtained within the range of 0.2-100.0 mg/L (r = 0.999). The relative recovery was between 97.82% and 101.20%, and the absolute recovery was between 84.37% and 88.80%. The intra-day and inter-day RSD was over 5%. The plasma concentration timedependent reliability of Paraquat was unimodal curve with the peak value obtained 2 h after administration, then the concentration of paraquat in serum decreased. The concentration of paraquat could not be detected after 24 h in the low-dosage group and after 72 h in the high-dosage group. Variance of repeated measurement analysis revealed significant differences in the concentrations of paraquat among different time points (F = 221.627, P = 0.000), different dosage groups (F = 133.618, P = 0.000), and change trends with time of different dosage groups (F = 81.874, P = 0.000), respectively. Conclusions The established HPLC can be used to determine the concentration of paraquat in rabbit serum. Our results showed that the paraquat concentration and its accumulation time are positively correlated with the dosage, and the concentration reaches the peak value 2 h after administration.