Centrins are a family of small calcium-binding proteins with diverse cellular functions that play an important role in centrosome biology. We previously identified Centrin 2 and Centrin 3 (Cetn2 and Cetn3) as substrates of the protein kinase Mps1. However, while Mps1 phosphorylation sites control the function of Cetn2 in centriole assembly and promote centriole over production, Cetn2 and Cetn3 are not functionally interchangeable, and we show here that Cetn3 is both a biochemical inhibitor of Mps1 catalytic activity and a biological inhibitor of centrosome duplication. In vitro, Cetn3 inhibits Mps1 autophosphorylation at Thr 676, a known site of T-loop autoactivation, and interferes with Mps1-dependent phosphorylation of Cetn2. The cellular overexpression of Cetn3 attenuates the incorporation of Cetn2 into centrioles and centrosome reduplication, while depletion of Cetn3 generates extra centrioles. Finally, overexpression of Cetn3 reduces Mps1 Thr 676 phosphorylation at centrosomes, and mimicking Mps1-dependent phosphorylation of Cetn2 bypasses the inhibitory effect of Cetn3, suggesting that the biological effects of Cetn3 are due to the inhibition of Mps1 function at centrosomes.