Ultraviolet B (UVB) induces morphological and functional changes of the skin. This study investigated the effect of UVB on keratinocyte senescence and the development of reconstructed human epidermis (RHE). Primary normal human keratinocytes (NHK) from juvenile foreskin were irradiated with UVB (30 mJ cm−2) and these effects were compared to NHK that underwent senescence in the late passage. UVB enhanced the accumulation of reactive oxygen species (ROS) and halted cell replication as detected by BrdU cell proliferation assay. The senescence phenotype was evaluated by beta-galactosidase (β-gal) staining and qPCR of genes related to senescent regulation,