Background: Since thrombolysis is the only approved intervention for ischemic stroke, improving its efficacy and safety is a therapeutic aim of considerable interest. The activated form of thrombin activatable fibrinolysis inhibitor (TAFI) has antifibrinolytic effects, and inhibition of TAFI might thus favor recanalization. The present study compared efficacy between TAFI inhibition alone and TAFI inhibition in combination with rtPA at a suboptimal dose, in a murine model of thromboembolic stroke. Methods: Focal ischemia was induced in mice by thrombin injection in the middle cerebral artery. Animals were placed within the magnet immediately after surgery for baseline MRI (H0). MRI examination comprised diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI), and T2-weighted imaging (T2-WI). Animals were randomly assigned to 1 of 5 treatment groups: saline, rtPA 5 mg/kg (tPA⁵: suboptimal or low dose), rtPA 10 mg/kg (tPA¹⁰: standard dose), TAFI-I 100 mg/kg (TAFI-I), and rtPA 5 mg/kg + TAFI-I 100 mg/kg (tPA⁵ + TAFI-I). Treatments were administered inside the magnet, via a catheter placed in the tail vein, using a power injector, as 10% bolus and 90% infusion over a period of 20 min. MRI examination was repeated at 3 h (H3) and 24 h (H24) after surgery. Therapeutic benefit was evaluated by: (1) improvement of reperfusion and (2) reduction in final lesion size. Microhemorrhages were assessed as black spots on T2-WI at H24. Animals were sacrificed after the last MR examination. The surgeon and all investigators were blinded to treatment allocation. Results: A total of 104 mice were operated on. Forty four of these were excluded from the study and 27 from the analysis, according to a priori defined criteria (no lesion or no mismatch), leading to the following distribution: saline (n = 6), tPA⁵ (n = 8), tPA¹⁰ (n = 7), TAFI-I (n = 7), and TAFI-I + tPA⁵ (n = 5). Standard-dose rtPA treatment (tPA¹⁰) significantly improved lesion regression between H0 and H24 compared to saline (-57 ± 18% vs. -36 ± 21%, p = 0.03), which treatment with rtPA⁵ or TAFI-I alone did not. On the other hand, combined treatment with tPA⁵ + TAFI-I showed only a trend toward lesion regression (-49 ± 26%), similarly to treatment with tPA¹⁰, but not significantly different from saline (p = 0.46). Nine animals showed microhemorrhage on T2-WI at H24. These animals were evenly distributed between groups. Conclusions: The present study showed that the combination of TAFI-I with a suboptimal dose of rtPA is not as effective as the standard dose of rtPA, while TAFI inhibition alone is not effective at all. The thromboembolic model is of particular interest in assessing rtPA association to improve thrombolysis, especially when coupled with longitudinal MRI assessment.