Poxviruses are large, enveloped, double-stranded DNA viruses. Vaccinia virus was used to eradicate smallpox and constitutes the prototypic poxvirus. The potential use of smallpox as a biological weapon, together with the high rate of adverse effects associated with vaccination have raised the need to find new therapeutic targets to treat poxvirus infections. During replication in the host cell cytoplasm, three different viral forms are generated: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular virions (EV). After their morphogenesis, a subset of IMV migrates to the trans-Golgi network and early endosome to acquire a two membrane wrapping and yield IEV. In contrast to IMV, which mainly remain inside the infected cell, IEV are transported on microtubules to the plasma membrane where fusion occurs to release EV. This viral form has the ability to promote actin polymerization beneath the plasma membrane facilitating cell-to-cell spread of the virus. While several viral proteins have been identified, the host cell machinery involved in viral actin-based motility is poorly understood. We combined the RNA interference technology, with high-throughput fluorescence microscopy and computer assisted image analysis to image cells infected with recombinant vaccinia virus that expresses a GFP-fusion membrane protein (vGFP). This system allowed us to monitor vaccinia virus spread in epithelial cells. These observations lay the basis to use the high-throughput siRNA screening approaches to identify novel cell factors required for the process of actin-dependent cell-to-cell transmission of vaccinia virus. Our systematic identification of host factors involved in viral cell-to-cell spread may reveal novel targets for therapeutic intervention.