Objective: B-cell specific moloney leukemia virus insertion site 1 (Bmi-1) has been extensively documented for its role in stem cell proliferation and differentiation, but its role in the brain of aged mice is unclear. The aim of this study was to investigate the pathophysiological role of Bmi-1 in brain aging. Methods: Seventeen-month-old Bmi-1 heterozygous (Bmi-1+/-) mice and wild-type (WT) mice were selected for behavioural observation and brain pathological analysis. Results: Bmi-1+/- mice showed diminished long-term spatial memory function, compared with WT mice (P < 0.05), accompanied by reductions in neurogenesis in the hippocampal dentate gyrus (DG) (P < 0.05), the neuronal number (P < 0.05), and the volume of grey matter areas (P < 0.05). Further studies revealed that neuronal mitochondria in the DG area of Bmi-1+/- mice had an increased proportion of inflated and swollen mitochondria with reduced cristae compared with WT mice (P < 0.05). The amount of lipofuscin in the cytoplasm of neurons in the DG area of Bmi-1+/- mice was significantly higher than that of WT mice (P < 0.05). Compared with WT mice, the mitochondrial energy metabolism-related proteins NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and NADH dehydrogenase ( ubiquinone) ferrithionein 3 (NDUFS3) were down-regulated in the DG region of Bmi-1+/- mice (P < 0.05), and Dihydrolipoyl transacetylase (DLST), an important catalysing enzyme of the tricarboxylic acid cycle, was also significantly down-regulated (P < 0.01), while Bmi-1 among the cell cycle factors regulated by Bmi-1, Cyclin-dependent kinase inhibitor p27 (p27) and Oncoprotein p53 (p53) were significantly up-regulated (P < 0.05). Conclusion: Half dose deletion of the Bmi-1 gene inhibits the production of newborn neurons in the hippocampal region of the aged mice, leading to a specific reduction in the volume of the hippocampal DG region and long-term memory dysfunction. The mechanism may be related to the abnormal expression of aging-related proteins p27 and p53 and neuronal mitochondrial degeneration.