Aim: To study the diagnostic and prognostic role of serum galectin-1 (Gal-1) and -3 (Gal-3) in acute ischemic stroke (AIS) patients. Methods: We enrolled 233 patients with first-ever acute ischemic stroke and 252 healthy controls in this study. The AIS severity was evaluated by National Institutes of Health Stroke Scale (NIHSS) scores. The serum Gal-1 and -3 levels were determined. All patients were followed for 1 years and the functional outcome were evaluated by modified Rankin Scale (mRS) scores. Results: We found that AIS patients had higher serum Gal-1 and -3 levels than controls. The serum Gal-3 level was closely associated with the AIS severity indicated by NHSS and infarction volume. Serum Gal-3 levels were significantly higher in patients with a poor outcome indicated by mRS scores than those in patients with a good outcome. In contrast, the serum Gal-1 is not associated with the severity and outcome of acute AIS patients. Our in vitro studies show that Gal-3 knockdown with siRNA dramatically increased the culture neuron cell viability and reduced apoptosis under oxygen glucose deprivation treatment. Meanwhile, the pro-inflammatory cytokine expression decreased with the inhibition of Gal-3. Conclusion: Our finding provides a novel biological marker, serum Gal-3, for monitor of acute AIS patients.