Abstract Chromosome condensation and segregation are highly orchestrated during mitosis. In early mitosis, dramatic structural changes occur to produce metaphase chromosomes, each consisting of a pair of compacted sister chromatids (condensation), At anaphase onset, a signal is produced to disrupt the linkage between sister chromatids (segregation), allowing them to be pulled apart to two daughter cells. Recently, we discovered that the MIIP protein, encoded by the Migration and invasion inhibitory protein gene located on chromosome 1p36, plays a critical role in mitotic transition through negatively regulating APC/CCdc20 activity and stabilizing cyclin B1 in glioma cells. Here we report that MIIP inhibits chromosome condensation and segregation resulting in G2/M arrest and polyploidy cell formation in colon cancer cells. Gain-of-function studies showed that the MIIP expression inhibited colony formation of isogenic HCT116 cells independent of the TP53 and PUMA status. To understand the potential role of MIIP in colon cancer cells, a panel of six colon cancer cell lines (DLD1, SW620, SW480, HCT116, NCI-747 and SW837) was used to elevate MIIP expression by adenovirus expression vector (Ad-MIIP). The G2/M arrest and polyploidy cell formation were observed in all the six cell lines we tested irrespective of the status of microsatellite stability and TP53 mutation. By using double thymidine block strategy, we synchronized HCT116 cells at G1/S boundary and released the cells in fresh medium to enrich the cell population in different stage during the cell cycle. We found that the expression of MIIP is cell cycle dependent and peaks at mitosis. Two major mitotic regulators, cyclin B1 and securin, were induced by MIIP. The cycloheximide (CHX) chase experiments showed that MIIP blocked cyclin B1 and securin degradation, which is consistent with our previous finding that MIIP directly interacted with Cdc20 and inhibited APC/CCdc20 activity. We further found that MIIP inhibited histone H3 phosphorylation at serine 10. Taken together, our studies provide evidence that MIIP plays a critical role in chromosome condensation and segregation in mitosis. Because chromosome 1p36 region is commonly deleted in colon cancer cells, the MIIP gene may represent a novel negative cell cycle regulator that is abnormally altered thus contributing to genomic instability in colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2011-3000