The formin family of proteins promotes the assembly of linear actin filaments in the cells of diverse eukaryotic organisms. The predominant formins in mammalian cells are self-inhibited by an intramolecular interaction between two terminal domains and are activated by the binding of the Rho GTPases and other factors. In this study, we show that Bni1p, a formin required for the assembly of actin cables in budding yeast, is also regulated by an autoinhibitory mechanism and phosphorylation by the actin regulatory kinase Prk1p, and possibly Ark1p as well, plays a key role in unlocking the inhibition. Bni1p is phosphorylated by Prk1p at three [L/V/I]xxxxTG motifs in vitro, and the phosphorylation is sufficient to activate Bni1p by disrupting its intramolecular interaction. This finding extends the roles of Prk1p in the regulation of actin dynamics to be associated with both anterograde and retrograde transport pathways, i.e. exocytosis and endocytosis, in yeast.