Preconditioning exercise prior to stroke exerts neuroprotection, which is an endogenous strategy that leads the brain cells to express several intrinsic factors and inhibits their apoptosis. However, it is unclear how long these benefits last after exercise cessation. The aim of this study was to investigate the effects of detraining on preconditioning exercise-induced neuroprotective potential after stroke. Rats were trained using a treadmill for aerobic exercise 5 days each week for 3 weeks, and their neuroprotective effects were examined until 3 weeks after exercise cessation. Stroke was induced by 60 min of left middle cerebral artery occlusion at 3 days, 1, 2, and 3 weeks after exercise cessation. Infarct volume, neurological deficits, sensorimotor function, expression levels of brain-derived neurotrophic factor (BDNF), hypoxia-induced factor-1α (HIF-1α), glial fibrillary acidic protein (GFAP), and P2X7 receptors, and apoptosis activity were examined using immunohistochemical and western blot analyses. Preconditioning exercise significantly reduced infarct volume and ameliorated sensorimotor function after stroke, and its beneficial effects were observed until 2 weeks after exercise cessation. The expression level of BDNF in the ischemic brain was significantly upregulated at 3 days after exercise cessation; however, the expression levels of HIF-1α, GFAP, and P2X7 receptor were significantly increased until 2 weeks after exercise cessation; thereby, significant anti-apoptotic effects were lost at 3 weeks of detraining. Our findings suggest that preconditioning exercise-induced neuroprotective potential may be lost shortly after exercise cessation. Neuroprotection through intrinsic protective factors, such as BDNF and HIF-1α, may provide different neuroprotective mechanisms in a time-dependent manner during detraining.