Toll-like receptor 9 (TLR9) agonists are being developed for treatment of colorectal and other cancers, yet the impact of these drugs on human intestines remains unknown. This, together with the fact that there are additional potential indications for TLR9 agonist therapy (eg, autoimmune and infectious diseases), led us to investigate the impact of MGN1703 (Lefitolimod) on intestinal homeostasis and viral persistence in HIV-positive individuals. Colonic sigmoid biopsies were collected (baseline and week four) from 11 HIV+ individuals on suppressive antiretroviral therapy, who received MGN1703 (60 mg sc) twice weekly for 4 weeks in a single-arm, phase 1b/2a study. Within sigmoid mucosa, global transcriptomic analyses revealed 248 modulated genes (false discovery rate< 0.05) including many type I interferon (IFN)-stimulated genes. MGN1703 increased the frequencies of cells exhibiting MX1 (P= 0.001) and ISG15 (P= 0.014) protein expression. No changes were observed in neutrophil infiltration (myeloperoxidase; P= 0.97). No systematic effect on fecal microbiota structure was observed (analysis of similarity Global R=− 0.105; P= 0.929). TLR9 expression at baseline was inversely proportional to the change in integrated HIV DNA during MGN1703 treatment (P= 0.020). In conclusion, MGN1703 induced a potent type I IFN response, without a concomitant general inflammatory response, in the intestines.