Abstract: The enantiomers of cyproconazole were separated by reverse-phase high-performance liquid chromatography using a chiral column Lux Cellulose-1 (cellulose-tris-(3,5-dimethylphenylcarba-mate),packed with 3 μm and 5 μm particles). The effects of different particle size, mobile phase composition and column temperature on the retention and resolution were investigated. Furthermore, the elution order of enantiomers was identified with an optical rotation detector. Moreover, the mechanism of enantiomeric separation was analyzed by measuring the thermodynamical parameters. The results showed that using acetonitrile-water as the mobile phase gave a better enantioseparation than the methanol-water system, and lower column temperature was more helpful for the separation. The ideal separation of cyproconazole on the 3 μm column at a flow rate of 0.3 mL/min and the 5 μm column at a flow rate of 1.0 mL/min could be achieved with V(acetonitrile):V(water)=60:40 at 20 ℃. When satisfactory enantioseparation was obtained, 3 μm column had the priority to be used and the elution order based on the optical rotation were(+/+/-/-). However, when the mobile phase changed from acetonitrile-water to methanol-water, the elution order would change to(+/-/+/-).Thermodynamic results show that the linear relationship of van't Hoff equation was good (R2 > 0.92) at the experimental temperature in the range of 5-40 ℃. The enantiomeric separation of cyproconazole was driven by enthalpy when using acetonitrile-water as the mobile phase. However, when using methanol-water as the mobile phase, the enantiomeric separation was driven by both enthalpy and entropy.