Cohesin is a highly conserved multi-subunit complex that holds sister chromatids together in mitotic cells. At the metaphase to anaphase transition, proteolytic cleavage of the α kleisin subunit (Rad21) by separase causes cohesin’s dissociation from chromosomes and triggers sister chromatid disjunction. To investigate cohesin’s function in post-mitotic cells, where it is widely expressed, we have created fruit flies whose Rad21 can be cleaved by TEV protease. Cleavage causes precocious separation of sister chromatids and massive chromosome missegregation in proliferating cells but not disaggregation of polytene chromosomes in salivary glands. Crucially, cleavage in post-mitotic neurons is lethal. In mushroom body neurons it causes defects in axon pruning while in cholinergic neurons it causes highly abnormal larval locomotion. These data demonstrate essential roles for cohesin in non-dividing cells, and also introduce a powerful new tool to investigate protein function in metazoa.