The present study provides a comparative understanding of florfenicol deposition kinetics following oral administration at a single dose of 30 mg/kg body weight to Japanese eel (Anguilla japonica) at the temperature of 20, 24 and 28 ℃, respectively. Approximate 0.3 mL of blood sample from each eel in these three groups was collected in a row at the time 0.5, 1, 2, 4, 8, 12, 24 and 36 h after medicated feed gavage. The concentrations of florfenicol in eel plasma were detected by reversed phase high performance liquid chromatography (RP HPLC). The plasma concentration time data of each eel were analyzed by non compartmental methods based on statistical moment theory using DAS software. Pharmacokinetics parameters of different groups were tested by one way analysis of variance using SPSS software. It was found that most of parameters were different significantly between groups (P<0.05). In the three groups at 20, 24 and 28 ℃, pharmacokinetics parameters of peak plasma concentration (Cmax) were (7.839±1.125), (13.010±2.334) and (18.267 ± 3.717) μg/mL, and the time to reach the Cmax (Tmax) were (6.500 ± 2.070), (4.500 ± 1.414) and (3.429 ± 0.926) h, respectively. These suggested that eels at higher temperature absorbed more drug and more quickly. The volumes of distribution (Vz/F) were (3.964±0.594), (2.466±0.672) and (1.841 ± 0.485) L/kg, respectively. The difference deduced that more florfenicol was bound to tissue in eels at lower temperature. The mean residence time (MRT0 ∞) and the half life of drug (t1/2z) in the three respective groups were (31.503 ± 7.117), (22.881 ± 4.940) and (22.134±6.204) h, and (21.243 ± 5.166), (14.994 ± 4.293) and (14.656 ± 5.061) h. These parameters showed that the elimination rate of florfenicol in eels at 24 ℃ or 28 ℃ was more quickly than that at 20 ℃. The areas under the concentration time curve (AUC0-∞) were (235.580 ± 62.013), (271.983 ± 75.023) and (353.192 ± 92.491) μg·h/mL, respectively. It indicated that the relative bioavailability of florfenicol was higher significantly in eels at higher temperatue.