Chemotherapy‐associated memory deficits in adults are prevalent with systemic treatment utilizing 5‐fluorouracil (5‐Fu). 5‐Fu disrupts cell proliferation and readily crosses the blood–brain barrier. Proliferating cells within the adult dentate gyrus of the hippocampus give rise to new neurons involved in memory and learning and require neurotrophic factors such as brain‐derived neurotrophic factor (BDNF) to nurture this process of adult neurogenesis. Some of these proliferating cells are anatomically and functionally supported by vascular endothelial cells. We propose that systemically administered 5‐Fu chemotherapy will cause deficits in hippocampal memory that are associated with altered BDNF levels and proliferating cells (particularly vascular‐associated cells) in the dentate gyrus. This was tested by determining the effect of 5‐Fu on spatial working memory as modelled by the object location recognition test. Numbers of vascular‐associated (VA) and non‐vascular‐associated (NVA) proliferating cells in the dentate gyrus were measured using double‐labelling immunohistochemistry with markers of proliferation (Ki67) and endothelial cells (RECA‐1). 5‐Fu‐induced changes in hippocampal BDNF and doublecortin (DCX) protein levels were quantified using Western immunoblotting. 5‐Fu chemotherapy caused a marginal disruption in spatial working memory and did not alter the total proliferating cell counts or the percentage of VA and NVA proliferating cells in the dentate gyrus. In contrast, 5‐Fu significantly reduced BDNF and DCX levels in the hippocampus, indicating alterations in neurotrophin levels and neurogenesis. These findings highlight the usefulness of animal models of ‘chemobrain’ for understanding the mechanisms that underlie chemotherapy‐associated declines in cognitive performance and memory.