Understanding the molecular basis for proper cell division requires a detailed functional analysis of microtubule (MT) associated proteins. MT associated protein 1S (MAP1S), the most ubiquitously expressed MAP1 family member, is required for accurate cell division. Using quantitative analysis of MT plus end tracking we show here that MAP1S knockdown alters MT dynamics throughout the cell cycle. Surprisingly, MAP1S downregulation results in faster growing, yet short-lived MT in all cell cycle stages and a global loss of MT acetylation. These aberrations correlate with severe defects in the final stages of cell division. In monopolar cytokinesis assays, we demonstrate that MAP1S guides MT dependent initiation of cytokinesis. Our data underline the key role of MAP1S as a global regulator of MT stability and demonstrate a novel primary function of MAP1S to regulate MT dynamics at cytokinesis onset.