Abstract: The conditioning method has an important influence on the compound-specific analysis results of hydrogen isotope ratios. The effects of three methods (injection of n-hexane or methane in the inlets, the reverse injection of methane) on the determination of hydrogen isotope values were investigated and compared. The results showed that the method of reverse injection of methane before each sample analysis can ensure the consistency of conditioning in the pyrolysis furnace tube, and can provide the best repeatability and accuracy of the compound-specific hydrogen isotope determination of natural gas.