Rejuvenation treatment to restore the juvenile characteristics of mature materials is an important approach to improve the adventitious rooting capacity of difficult-to-root woody plants. However, the in-deep mechanism is current less well defined. In this study, walnut young and young cuttings were used as materials. Through methylation modification-dependent endonuclease sequencing technology (MethylRAD-Seq), DNA methylation in young and young cuttings was detected at the genome-wide level. The site distribution characteristics further analyzed the expression of differentially methylated site-related genes in cuttings before and after rejuvenation. The results showed that: rejuvenation treatment could significantly reduce the level of DNA methylation in walnut cuttings; functional enrichment analysis showed that genes related to differential methylation sites were mainly involved in brassinolide signal transduction, secondary metabolite biosynthesis, and Lignin biosynthesis and other functions, participate in metabolic pathways such as photosynthesis, MAPK signaling pathway, fructose and mannose metabolism, cAMP signaling pathway and phenylpropane biosynthesis; qRT-PCR analysis results show that key regulatory genes occur in adventitious roots before and after rejuvenation NAC1, ARF5, ARF6 and WRKY22 have different expression patterns in rejuvenated and adult materials. It suggested that the rejuvenation treatment reduced the genomic DNA methylation level in walnut cuttings, and then affected the expression of key functional genes in adventitious root formation. It may be an important way for rejuvenation to regulate adventitious root formation in walnut.