The intestinal innate immune system continually interacts with commensal bacteria, thus oral vaccines should induce extra/alternative activation of DC, potentially through TLR. To examine this we collected intestinal lymph DC (iL-DC) under steady-state conditions and after feeding resiquimod (R-848), a synthetic TLR7/8 ligand, which we showed induces complete emptying of gut DC into lymph. iL-DC are heterogeneous with subset-specific functions. In this study we determined the kinetics of iL-DC subset release, activation and cytokine secretion induced by R-848. We show that L-DC comprise three distinct subsets (CD172ahigh, CD172aint and CD172alow) present with similar frequencies in intestinal but not hepatic lymph. No iL-DC express TLR7 mRNA, and only CD172a+ iL-DC express TLR8. However, after oral R-848 administration, output of all three subsets increases dramatically. CD172ahigh DC release precedes that of CD172alow DC, and the increased frequency of CD25high iL-DC is restricted to the two CD172a+ subsets. After feeding R-848 only CD172ahigh iL-DC secrete IL-6 and IL-12p40. However, CD172aint and CD172ahigh DC secrete similar but markedly lower amounts when stimulated in vitro. These results highlight the importance of in vivo approaches to assess adjuvant effects on DC and give novel insights into the subset-specific effects of an oral TLR ligand on intestinal DC.