Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking site for the Aurora B complex at centromeres. Aurora B functions to correct improper kinetochore-microtubule attachments and alert the spindle checkpoint to the presence of misaligned chromosomes. We show that Haspin inhibitors decreased H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of centromere and kinetochore Aurora B substrates. Consequently, metaphase chromosome alignment and spindle checkpoint signaling were compromised. These effects were phenocopied by microinjection of anti-H3T3ph antibodies. Retargeting Aurora B to centromeres partially restored checkpoint signaling and Aurora B-dependent phosphorylation at centromeres and kinetochores, bypassing the need for Haspin activity. Haspin inhibitors did not obviously affect phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed. Haspin inhibitors did not block Aurora B localization to the spindle midzone in anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal centromeric roles of Aurora B in chromosome movement and spindle checkpoint signaling.