Dendritic cell (DC)-based vaccines are a promising strategy for tumor immunotherapy due to their ability to activate both antigen-specific T-cell immunity and innate immune effector components, including natural killer (NK) cells. However, the optimal mode of antigen delivery and DC activation remains to be determined. Using M protein mutant vesicular stomatitis virus (DeltaM51-VSV) as a gene-delivery vector, we demonstrate that a high level of transgene expression could be achieved in approximately 70% of DCs without affecting cell viability. Furthermore, DeltaM51-VSV infection activated DCs to produce proinflammatory cytokines (interleukin-12, tumor necrosis factor-alpha, and interferon (IFN)alpha/beta), and to display a mature phenotype (CD40(high)CD86(high) major histocompatibility complex (MHC II)(high)). When delivered to mice bearing 10-day-old lung metastatic tumors, DCs infected with DeltaM51-VSV encoding a tumor-associated antigen mediated significant control of tumor growth by engaging both NK and CD8(+) T cells. Importantly, depletion of NK cells completely abrogated tumor destruction, indicating that NK cells play a critical role for this DC vaccine-induced therapeutic outcome. Our findings identify DeltaM51-VSV as both an efficient gene-delivery vector and a maturation agent allowing DC vaccines to overcome immunosuppression in the tumor-bearing host.