Aurora B, the catalytic subunit of the chromosomal passenger complex (CPC), is a key regulator of many processes during mitosis and cytokinesis. Aurora B activation is controlled by the CPC member INCENP, which serves as a regulatory target to fine-tune Aurora B kinase activity. Now, Sandrine Ruchaud and colleagues (Papini et al., 2019) characterise a novel motif within INCENP, termed the STD motif. Using phosphoproteomic analysis, they identify three conserved residues in the IN-box of INCENP, the domain responsible for INCENP binding to Aurora B, and generate INCENP mutants that mimic or prohibit phosphorylation at these sites in an INCENP conditional knockout chicken cell line. Remarkably, a S752A/T753A (INCENPST752AA) mutant behaves as an INCENP-null. It and, to a lesser extent, the S752E/T753E (INCENPST752EE) mutant display reduced Aurora B activity compared to wild-type INCENP. Accordingly, both mutants show chromosome alignment and spindle checkpoint defects, but surprisingly, only INCENPST752AA displays defects in cytokinesis. INCENPST752EE instead causes the formation of ectopic cleavage furrows, even in interphase and early mitosis, and this depends on the kinase activity of Aurora B, ROCK1 and Plk1. Plk1 is also involved in the phosphorylation of INCENP at T753, although this requires a priming phosphorylation at S752. Thus, this work shows that phosphoregulation of the newly-identified STD motif within INCENP is critical for correct chromosome alignment and timing of cytokinesis.