Rhodococcussp. strain CH91 is capable of utilizing long-chainn-alkanes as the sole carbon source. Two new genes (alkB1andalkB2) encoding AlkB-type alkane hydroxylase were predicted by its whole-genome sequence analysis. The purpose of this study was to elucidate the functional role ofalkB1andalkB2genes in then-alkane degradation of strain CH91. RT-qPCR analyses revealed that the two genes were induced byn-alkanes ranging from C16 to C36 and the expression of thealkB2gene was up-regulated much higher than that ofalkB1. The knockout of thealkB1oralkB2gene in strain CH91 resulted in the obvious reduction of growth and degradation rates on C16-C36n-alkanes and thealkB2knockout mutant exhibited lower growth and degradation rate than thealkB1knockout mutant. When genealkB1oralkB2was heterologously expressed inPseudomonas fluorescensKOB2Δ1, the two genes could restore its alkane degradation activity. These results demonstrated that bothalkB1andalkB2genes were responsible for C16-C36n-alkanes’ degradation of strain CH91, andalkB2plays a more important role thanalkB1. The functional characteristics of the twoalkBgenes in the degradation of a broad range ofn-alkanes make them potential gene candidates for engineering the bacteria used for bioremediation of petroleum hydrocarbon contaminations.