Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MV are further wrapped and secreted to spread infection within the host. However, late during infection MV accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titres. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate-limiting for EV production. The A26 gene is conserved within vertebrate poxviruses, but strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfil different functions represents a novel level of complexity in mammalian viruses with major impact on evolution, adaptation and transmission.