Following an oral inoculation,descends to the mouse large intestine for long-lasting colonization. However, a mutantthat lacks the plasmid-encoded protein pGP3 due to an engineered premature stop codon (designated as CMpGP3S) failed to do so even following an intrajejunal inoculation. This was because a CD4T cell-dependent immunity prevented the spread of CMpGP3S from the small intestine to the large intestine. In the current study, we found that mice deficient in IL-22 (IL-22) allowed CMpGP3S to spread from the small intestine to the large intestine on day 3 after intrajejunal inoculation, indicating a critical role of IL-22 in regulating the chlamydial spread. The responsible IL-22 is produced by CD4T cells since IL-22mice were rescued to block the CMpGP3S spread by donor CD4T cells from C57BL/6J mice. Consistently, CD4T cells lacking IL-22 failed to block the spread of CMpGP3S in Rag2mice, while IL-22-competent CD4T cells did block. Furthermore, mice deficient in cathelicidin-related antimicrobial peptide (CRAMP) permitted the CMpGP3S spread, but donor CD4T cells from CRAMPmice were still sufficient for preventing the CMpGP3S spread in Rag2mice, indicating a critical role of CRAMP in regulating chlamydial spreading, and the responsible CRAMP is not produced by CD4T cells. Thus, the IL-22-producing CD4T cell-dependent regulation of chlamydial spreading correlated with CRAMP produced by non-CD4T cells. These findings provide a platform for further characterizing the subset(s) of CD4T cells responsible for regulating bacterial spreading in the intestine.