Replication-defective recombinant Adenoviruses are the most widely studied replication-defective vectors for the potential treatment of inherited human diseases. However, broad clinical application of replication-defective Adenoviruses in gene therapy is being hindered by the induction of vigorous innate and adaptive immune responses against the vector which causes deleterious effects in the liver. Valpha14 invariant natural killer T (Valpha14iNKT) cells are thymic-derived innate T cells at the interface between the two arms of the immune response and provide full engagement of host defense. The pathophysiological role of intrahepatic Valpha14iNKT cells during replication-defective Adenovirus infection is not known, and is the main focus of our study. Our data showed that intrahepatic Valpha14iNKT cells were activated in response to Adenovirus infection to induce significant levels of hepatic CCL5 and subsequent liver toxicity. Moreover, intrahepatic CCL5 production was selectively reduced by Valpha14iNKT cell deficiency. In vivo studies utilizing CCL5 deficient mice or Valpha14iNKT cell deficient mice demonstrated that CCL5 or Valpha14iNKT cell deficiency were each associated with reduced liver pathology. Similar results were seen after blocking the biological effects of the CCL5 receptors. In conclusion, we have identified an important pro-inflammatory role for activated intrahepatic Valpha14iNKT cells in positively influencing hepatic CCL5 production to promote acute liver inflammation and injury. Therefore, our findings highlight the blockade of CCL5 interaction with cognate receptor(s) as an important potential strategy to alleviate liver pathology associated with replication-defective Adenovirus infection.