Accumulating evidence suggests that chronic metformin preconditioning offers potent neuroprotective effects against ischemic stroke. However, the underlying mechanisms remain largely unknown. In this study, we tested the hypothesis that chronic preconditioning with metformin conferred neuroprotection via suppression of nuclear factor kappa B (NF-κB)-mediated inflammatory pathway. Male Sprague-Dawley rats were treated with vehicle or metformin (50 mg/kg daily, i.p.) for 3 weeks and were subjected to permanent middle cerebral artery occlusion (pMCAO). At 24 h (acute phase) and 96 h (subacute phase) after pMCAO, infarct volume and neurological deficits were evaluated. Meanwhile, the activity of NF-κB and the levels of its downstream pro-inflammatory cytokines were detected at 24 h after pMCAO. Our results showed that chronic metformin preconditioning significantly reduced infarct volume and improved neurological deficits at 24 and 96 h after pMCAO. It also suppressed brain NF-κB activity, which was accompanied by a reduction of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and induced nitric oxide synthase in the peri-infarct regions at 24 h after pMCAO. Moreover, the microgliosis and astrocytosis induced by pMCAO were also ameliorated by chronic metformin preconditioning. Collectively, the present study provides the first evidence that suppression of NF-κB-mediated inflammatory pathway may represent one potential mechanism underlying the neuroprotection of chronic metformin preconditioning. In addition, our findings suggest that metformin, a first-line drug for glycemic control, has a practical clinical use for stroke prevention and treatment.