Human somatic cells can be reprogrammed to pluripotent stem cells by small molecules through an intermediate stage with a regeneration signature, but how this regeneration state is induced remains largely unknown. Here, through integrated single-cell analysis of transcriptome, we demonstrate that the pathway of human chemical reprogramming with regeneration state is distinct from that of transcription-factor-mediated reprogramming. Time-course construction of chromatin landscapes unveils hierarchical histone modification remodeling underlying the regeneration program, which involved sequential enhancer recommissioning and mirrored the reversal process of regeneration potential lost in organisms as they mature. In addition, LEF1 is identified as a key upstream regulator for regeneration gene program activation. Furthermore, we reveal that regeneration program activation requires sequential enhancer silencing of somatic and proinflammatory programs. Altogether, chemical reprogramming resets the epigenome through reversal of the loss of natural regeneration, representing a distinct concept for cellular reprogramming and advancing the development of regenerative therapeutic strategies. Display omitted •Human chemical reprogramming activates regeneration gene program•Epigenome remodeling of regeneration gene program with enhancer recommissioning•Chemically resetting epigenome through reversal of the loss of natural regeneration•Regeneration program activation requires silencing somatic and inflammatory programs By performing single-cell transcriptomic and histone modification profiling of the chemical reprogramming process, Wang et al. unveil the epigenome remodeling underlying activation of the regeneration program. They discover that sequential enhancer recommissioning and promoter activation mirrors a reversed pathway of regeneration loss in organism maturation and demonstrate the intrinsic barriers to this process.