Poxviruses, the largest mammalian viruses, encode two dual-specificity enzymes, F10 kinase and H1 phosphatase. These enzymes are essential signaling mediators during poxvirus infection. Yet their precise functional role in reversible protein phosphorylation during the virus lifecycle remains enigmatic. Using a phosphoproteomic strategy in combination with F10 and H1 inducible viruses, we established the F10/H1 dependent viral signal network in HeLa infected cells by the prototypic poxvirus, vaccinia. In order to relate defined perturbations of this signaling network to a phenotypic outcome, we performed a detailed characterization of the progeny virions’ proteotype. Our phosphoproteomic strategy allowed us to pinpoint relevant phosphorylation sites dependent on F10 and H1 which guided us towards focused functional analysis of two specific factors, the I7 protease required for proteolytic processing during virion maturation, and the viral early transcription factor, A7, showing that virion morphogenesis and the transcriptional competence of newly assembled virions are directly dependent on reversible phosphorylation of S134 in I7 and Y367 in A7. These results establish the molecular mode of action and a key role of F10/H1 in dynamic phospho-regulation during multiple stages of infectious poxvirus assembly.