Post-mitotic cell separation is one of the most prominent events in the life cycle of eukaryotic cells, but the molecular underpinning of this fundamental biological process is far from being concluded and fully characterized. We use budding yeast Saccharomyces cerevisiae as a model and demonstrate AMN1 as a major gene underlying post-mitotic cell separation in a natural yeast strain, YL1C. Specifically, we define a novel 11-residue domain by which Amn1 binds to Ace2. Moreover, we demonstrate that Amn1 induces proteolysis of Ace2 through the ubiquitin proteasome system and in turn, down-regulates Ace2's downstream target genes involved in hydrolysis of the primary septum, thus leading to inhibition of cell separation and clumping of haploid yeast cells. Using ChIP assays and site-specific mutation experiments, we show that Ste12 and the a1-α12 heterodimer are two direct regulators of AMN1. Specifically, a1-α2, a diploid-specific heterodimer, prevents Ste12 from inactivating AMN1 through binding to its promoter. This demonstrates how the Amn1-governed cell separation is highly cell type dependent. Finally, we show that AMN1368D from YL1C is a dominant allele in most strains of S. cerevisiae and evolutionarily conserved in both genic structure and phenotypic effect in two closely related yeast species, K. lactis and C. glabrata.