Background Intraportal administration of alloantigen is reported to reduce antigen-specific immune responses, although the underlying mechanisms for the reduced immunological reactions, especially those of the graft, are poorly understood. We examined intracellular cytokine production by graft-infiltrating lymphocytes (GILs) and peripheral blood lymphocytes (PBLs) to elucidate the underlying mechanisms of beneficial effects on intraportal infusion of donor cells in rat small bowel transplantation (SBT). Methods Recipient rats (Lewis) were transplanted with small bowel from ACI rats. Tacrolimus (Tac) was injected daily from days 0 to 4. Bone marrow cells of ACI rats were infused via the portal or tail vein on the day of surgery. On day 5, both GILs and PBLs collected from SBT graft and peripheral blood, respectively, were analyzed for intracellular cytokine production of recipient-derived αβ-T cells. The Th1/Th2 balance in each group was designated as the ratio of the percentage of GILs or PBLs staining positive for intracellular IL-4 or IFN-gamma, respectively. The total cell numbers of GILs from SBT graft were also counted. Results Survival of recipients was markedly prolonged by the combination of Tac and donor-specific bone marrow infusion via the portal vein (DS BMI-PV-Tac) compared with the untreated control, Tac alone, or DS BMI tail vein plus Tac. DS BMI-PV-Tac significantly decreased the total cell numbers of GILs and also induced remarkable Th2-type response in GILs. Conclusions Our results indicate that DS BMI-PV-Tac decreased GILs and enhanced Th2-type response in SBT graft, both of which are associated with a significant prolongation of graft survival in SBT.