Osteopontin (OPN) is a multifunctional protein which has shown neuroprotective properties in animal models of cerebral ischemia. Nevertheless, its role in acute human stroke has not yet been established. Therefore, we aimed to determine human serum OPN level during acute ischemic stroke and its relationship with patient outcome. We measured OPN levels in 178 consecutive patients with a middle cerebral artery (MCA) occlusion who received fibrinolytic therapy and in 40 control subjects. OPN level was determined by an enzyme-linked immunosorbent assay (ELISA). Bad functional outcome was defined by modified Rankin Scale (mRS) score >2 at 3 months after stroke onset. A logistic regression analysis was performed to determine factors that could be independently associated with poor prognosis. OPN levels among stroke patients did not differ from the controls' OPN levels (16.65 vs. 17.83 ng/mL, p = 0.404). Interestingly, OPN level was increased among those patients who showed worse prognosis at 3 months (19.96 vs. 15.48 ng/mL, p = 0.040). In a logistic regression model, an OPN level >27.22 ng/mL was found to be an independent factor for a bad outcome (OR 5.01, 95% CI 1.60-15.72, p = 0.006) after adjusting for potential confounders. Those patients showing higher OPN levels before tPA administration displayed a worse prognosis compared to those with lower OPN levels. Further research is necessary to elucidate the role of OPN in ischemic stroke pathophysiology and validate OPN as a useful tool to predict long-term stroke outcome.