Helper-dependent adenoviral vectors (HDAd) are effective tools for liver-directed gene therapy because they can mediate long-term transgene expression in the absence of chronic toxicity. However, high vector doses required for efficient hepatocyte transduction by intravascular delivery result in systemic vector dissemination and dose-dependent activation of the innate immunity. Therefore, strategies to achieve high-efficiency hepatocyte transduction using low vector doses and/or to reduce the acute elevations of proinflammatory cytokines and chemokines may have significant clinical potential. Vasoactive intestinal peptide (VIP) is an endogenous neuropeptide involved in the regulation of hepatic blood flow and plays an important role as modulator of immune functions. Here, we show that VIP pretreatment in mice is able to increase hepatocyte transduction by HDAd, decrease vector uptake by the spleen, reduce elevation of proinflammatory serum cytokines interleukin (IL)-6 and IL-12, and reduce serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) following intravenous HDAd injection. VIP pretreatment also resulted in a reduction in the expression of the chemokines macrophage-inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1 (MCP-1), and regulated on activation normal T-cell expressed and secreted (RANTES) in the livers of mice injected with HDAd. These results suggest that VIP can improve the therapeutic index of HDAd by increasing hepatocyte transduction efficiency while reducing cytokine and chemokine expression following intravascular delivery of HDAd.