Alternatively activated macrophages (AAMs) (or M2a) can inhibit colitis but may also be associated with fibrosis. Thus, by using the dinitrobenzene sulfonic (DNBS) murine model of colitis, this study aimed to determine whether 1) bone marrow (BM)-derived AAMs could reduce colitis, 2) any anticolitic effect of BM-AAMs was IL-10 dependent, and 3) repeated AAM treatments remained effective and were associated with fibrosis in the gut or other tissues. Balb/c mice received AAMs (10⁶ intraperitoneally) from wild-type (WT) or IL-10^−/− mice 48 h prior to DNBS (3 mg intrarectally) with disease assessed 72 h later, or they received three doses of DNBS at 2-wk intervals ± AAMs 6 h post-DNBS to mimic a treatment regimen. DNBS-treated mice developed colitis; this was significantly less severe in mice receiving WT AAMs and less so in animals given IL-10^−/− AAMs, indicating a role for IL-10 in the inhibition of DNBS-driven colitis. Similarly, after the third AAM treatment lesser colonic histopathology was observed compared with time-matched DNBS-only-treated animals, and notably there was no evidence of increased fibroses in the colon, terminal ileum, lung, or liver of AAM-treated mice as assessed by quantitative PCR for prolyl-4-hydrolase, α-smooth muscle actin, and collagen (type IIIα) and histochemical and biochemical assessment of collagen deposition. This study provides mechanistic insight to the anticolitic capacity of AAMs and indicates that repeated adoptive transfer of ex vivo programmed BM-AAMs is safe and efficacious in the treatment of DNBS-induced murine colitis, providing additional support for their consideration as an immunotherapy.