Cerebral ischemic disease is the most common cerebrovascular disease, especially ischemic stroke. Exercise has a protective function on brain tissues following cerebral ischemia-reperfusion injury (CIRI), but its preventive effect and mechanism in CIRI remain unclear. This research aimed to investigate the effect and mechanism of exercise preconditioning on CIRI. The middle cerebral artery occlusion (MCAO) operation was prepared to set up CIRI rats. All rats were randomized into the MCAO, exercise (exercise preconditioning plus MCAO operation), vector (exercise preconditioning, MCAO operation plus intraventricular injection of empty vector), and tissue inhibitor of metalloprotease 1 overexpression (OE-TIMP1) groups (exercise preconditioning, MCAO operation plus intraventricular injection of OE-TIMP1). The results indicated that exercise preconditioning effectively suppressed the brain dysfunction and TIMP1 mRNA level in MCAO rats, which was partially offset by OE-TIMP1. Also, the attenuation of exercise on the neuron death status and the infarction size in MCAO rats was counteracted by OE-TIMP1. This study also confirmed that exercise weakened apoptosis and oxidative stress damage, with a notable increase of Bcl-2, superoxide dismutase, and glutathione peroxidase production, and an evident decrease of Bax, caspase 3, and malondialdehyde in MCAO rats, while the effect was partially reversed after overexpressing TIMP1. Additionally, the down-regulation of exercise on the protein levels of TIMP1, hypoxia-inducible factor-alpha, vascular endothelial growth factor receptor-2, VEGF, and Notch 1 in MCAO rats was partially reversed by OE-TIMP1. Altogether, exercise preconditioning had protective effects on CIRI by restraining TIMP1. This study provides new targets and therapeutic strategies for the prevention of CIRI.