•Gene expression profiles of crypt and villus early after IR were characterized.•Activated P53, DNA damage, and apoptosis in stem cells/progenitors.•Transit amplifying cells retained Ki67 expression with less P53 activation and apoptosis. Cells respond differently to DNA damaging agents, which may related to cell context and differentiation status. The aim of present study was to observe the cellular and molecular responses of cells in different differentiation status to ionizing irradiation (IR). Crypt-villus unit of murine small intestine was adopted as a cell differentiation model. DNA damage responses (DDRs) of crypt and villus were observed 1–24h after 12Gy IR using gene expression microarray analysis, immunohistochemical staining, Western blotting and Electrophoretic Mobility Shift Assay. Microarray analysis revealed that most differentially expressed genes were related to p53 signaling pathway in crypt 4h after IR and in both crypt and villus 24h after IR. In crypt stem cells/progenitor cells, H2AX was phosphorylated and dephosphorylated quickly, Ki67 attenuated, cell apoptosis enhanced, phosphorylated P53 increased and translocated into nuclear with the ability to bind p53-specific sequence. In upper crypt (transit amplifying cells) and crypt-villus junction, cells kept survive and proliferate as indicated by retained Ki67 expression, suppressed p53 activation, and rare apoptosis. DDRs varied with cell differentiation status and cell function in small intestinal epithelium. P53 signaling pathway could be an important regulatory mechanism of DDRs.