Radiation-induced acute intestinal toxicity remains a major limitation to the delivery of tumoricidal doses of colorectal irradiation. Recent reports indicate that Toll-like receptor (TLR) agonists TLR4 and TLR5 protect against toxicity due to intestinal irradiation. The phenotype (M1 or M2) of macrophages expressing TLRs may play a role in tissue repair. The aim was to investigate whether administration of TLR4 agonist lipopolysac-charide (LPS) or TLR5 agonist flagellin after irradiation modified the recruitment and phenotype of colonic macrophages and improved tissue damage. Rats were exposed to single 20- or 27-Gy doses of colorectal irradiation. TLR4 agonist LPS or TLR5 agonist flagellin (at 50 or 200 μg/rat) was administered i.p. 3 days after irradiation. Flow cytometric analysis, immunostaining, and real-time polymerase chain reaction analysis were used to assess the M1/M2 phenotype and crypt cell proliferation 7 days after irradiation. Irradiation (20 and 27 Gy) increased TLR41 and TLR5+ macrophage frequency in the mucosa. LPS or flagellin administration maintained this elevated frequency after the 27-Gy irradiation. LPS and flagellin drove macrophages toward the anti-inflammatory M2 phenotype by increasing Arg1 and CD163 expression and microenvironmental effector molecules (C-C motif chemokine 22, transforming growth factor-β1, and interleukin-10). Proliferating cell nuclear antigen immunostaining, Ki67 expression, and antimicrobial factor Reg3γ showed that the M2 shift correlated with epithelial regeneration. In conclusion, administration of either LPS or flagellin after colorectal irradiation may provide effective protection against epithelial remodeling. This tissue repair was associated with an M2 macrophage shift. Using TLR agonists to moderately activate innate immunity should be considered as a strategy for protecting healthy tissue from irradiation. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.