Major histocompatibility complex-II (MHC-II) plays an important role in graft rejection and class II transactivator (CIITA) is the key regulator for MHC-II expression. The aim of this study was to determine the efficacy of intragraft inhibition of CIITA in attenuating liver transplant rejection. Three plasmids containing small hairpin RNA (shRNA) against rat CIITA (pCIITA-shRNA) and one control plasmid of pHK-shRNA were constructed. In vitro dendritic cell (DC) transfection and liver transfection via portal vein in donor rats (n = 8) by shRNA plasmids were performed to confirm the inhibitory effect of pCIITA-shRNA on CIITA expression. It showed that expressions of CIITA and MHC-II were significantly inhibited by pCIITA-shRNA in both DC in vitro and liver of donor rats in vivo (p < 0.05 vs. control pHK-shRNA treatment). pCIITA1-shRNA was proved to be the best inhibitor among three pCIITA-shRNAs and then used in high-responder rat liver transplantation model (DA donors-to-Lewis recipients). Transplant groups (n = 16/group) include untreated recipients transplanted with donor liver graft pretreated with either saline, or pHK-shRNA, or pCIITA1-shRNA. Cyclosporine-treated (10 mg/kg, im, day 0–7) recipients transplanted with unmodified liver grafts were used as no rejection control. The results showed that the recipient rats survived significantly longer in pCIITA1-shRNA-treated group with markedly attenuated liver graft rejection (p < 0.05 vs. saline and pHK-shRNA-treated groups). Furthermore, significantly decreased intragraft expressions of CIITA, MHC-II, IL-2, and IFN-γ were found in pCIITA1-shRNA-treated group (p < 0.05 vs. saline and pHK-shRNA-treated groups). This study suggests that intragraft inhibition of CIITA could be a novel strategy for attenuating graft rejection in liver transplantation. © 2014 Wiley Periodicals, Inc. Microsurgery 35:52–59, 2015.