Abstract Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event, involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 binding stimulates Plk4 kinase activity in vitro, and, in turn, is phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STAN domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N–terminus of Ana2 which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.