In order to comprehensively understand the genetic background of Paenarthrobacter ilicis CR5301 and dissect the key enzymes of CR5301 for transforming rebaudioside C (RC), the whole genome of CR5301 was sequenced using nextgeneration Illumina HiSeq and third-generation Nanopore-based DNA sequencing, and the key enzymes for RC convertion were predicted. The results showed that CR5301’s genome was a closed circular chromosomal DNA molecule without plasmids. The genome sequence was 4748281 bp in length, with a GC content of 62.92%. The genome encoded a total of 4458 genes, including 4 gene islands, 1 prophage and 14 CRISPR-Cas coding sequences. CR5301 is the first strain of P. ilicis whose genome has been completely determined, and it is also the strain with the largest known genome of the genus Paenarthrobacter. P. ilicis CR5301 was more closely related to P. aurescens, but more distantly related to P. ureafaciens, according to genome collinearity and 16S rRNA gene-based phylogenetic tree analysis. Comprehensive annotation analysis against seven protein databases showed that P. ilicis CR5301’s genome contained 523 carbohydrate active enzyme genes, of which 18 glycosidase genes may be the key enzyme genes for the transformation of RC by CR5301. Finally, the physicochemical properties and secondary structures of the 18 glycosidases were predicted by the bioinformatics software ProtParam and SOPMA. These results provide clear and complete genetic information for understanding the mechanism of RC transformation by CR5301, and provide a complete and reliable reference genome sequence for extensive biological studies on P. ilicis.