The key mitotic regulator pituitary tumor transforming gene (PTTG) is expressed at low levels in fetal brain compared with adult, and modulates the proliferation of human embryonic neuronal N-Tera2 (NT2) cells. We examined the function and expression of PTTGs interacting partner separase, along with Rad21, the functional component of cohesin, which is cleaved by separase following interaction with PTTG. In contrast to PTTG, the cleaved forms of separase and Rad21 were highly expressed in human fetal cerebral cortex compared with adult brain. All samples were collected with informed consent and ethical approval. In a murine model of absent PTTG expression the PTTG knock-out mouse separase and Rad21 were over-expressed in multiple brain regions. cDNA array analysis of other key mitotic regulators additionally identified cyclin C and sestrin 2 to be induced in the brains of PTTG-null mice compared with wild type. Further, Rad21 mRNA expression was highly correlated with that of PTTG, separase, cyclin C and sestrin 2 in fetal brains, but not in adult brains. In embryonic neuronal NT2 cells, siRNA repression of PTTG protein expression resulted in increased levels of the activated forms of Rad21 and separase, and promoted cell proliferation. siRNA depletion of separase protein expression, however, failed to alter PTTG or Rad21 levels, or cell turnover. Taken together, these data provide a novel insight into the expression and function of the mitotic regulators PTTG, separase and Rad21 in in vitro and in vivo models of neural cell growth, and suggest that modulation of PTTG expression in particular has profound implications for cell growth.