Continuous intravenous infusion (IV) or subcutaneous injection (SC) of insulin was widely applied to control hyperglycemia after ischemic stroke. However, the impact of different administration modes on glycemic variability was unknown. Consecutive stroke patients treated with intravenous thrombolysis were screened. Subjects who received insulin treatment were included and entered into the IV or SC group according to the respective administration mode. Blood glucose was closely monitored within the first 72 hours, and the target range of glucose was from 7.7 to 10.0 mmol/L for all patients. The variabilities of glucose, assessed using standard deviation of the mean, variable coefficient and range from the maximum to the minimum value, were compared between the two groups. A total of 130 patients were enrolled with 66 in the IV groups and 64 in the SC group. Compared with the SC group, the IV group had higher glycemic variability evaluated as either standard deviation (2.7 ± 0.7 mmol/L vs 2.2 ± 0.9 mmol/L, p = 0.002), variable coefficient (0.26 ± 0.06 vs 0.23 ± 0.08, p = 0.011) or range (10.0 ± 3.6 mmol/L vs 8.1 ± 3.1 mmol/L, p = 0.001). Multivariate logistic regression analyses found that continuous intravenous infusion was associated with higher level of the standard deviation (adjusted OR 3.01, 95% CI 1.29-7.28, p = 0.011), variable coefficient (adjusted OR 5.97, 95% CI 2.55-13.96, p < 0.001) and range (adjusted OR 6.08, 95% CI 2.63-14.05, p < 0.001). Continuous intravenous infusion of insulin was associated with higher glycemic variability than subcutaneous injection in acute stroke patients receiving thrombolysis.