Mannose-binding lectin (MBL), an initiator of the lectin pathway (LP) of complement activation, is detrimental in ischemic stroke, as shown in clinical studies and rodent models. Whereas humans have one functional MBL protein, rodents have two isoforms, MBL-A and MBL-C, whose functions relative to that of human MBL are unknown. To permit the clinical translation of preclinical data, we aimed to define the specific contributions of MBL-A and MBL-C to brain ischemia. We subjected mice with double (MBL-/-) or single (MBL-A-/- or MBL-C-/-) MBL isoform depletion to transient middle cerebral artery occlusion (tMCAo). MBL-/- mice had fewer neurological deficits and smaller ischemic lesions than WT mice. MBL-A-/- mice had smaller lesions than WT mice and exhibited no significant behavioral defects, whereas MBL-C-/- mice did not differ from WT mice. The induction of Mbl1 and Mbl2 (the MBL-A and MBL-C genes) expression 48 h after tMCAo was similar across genotypes. The time course of Mbl1 and Mbl2 expression in WT ischemic mice showed that Mbl1 activation occurred earlier (24 h) than Mbl2 activation (48 h). The plasma levels of MBL-A and MBL-C in MBL-C-/- and MBL-A-/- mice, respectively, were similar to those in WT mice both at baseline and at 48 h after tMCAo. At 48 h, MBL-A-/- ischemic mice showed higher MBL-C levels in the brain than WT mice. WT and MBL-C-/- ischemic mice had higher LP activity in plasma and, accordingly, higher levels of C3 deposition in the brain than MBL-A-/- and MBL-/- mice. In conclusion, mice with depletion of both MBL isoforms exhibited strong protection from ischemia/reperfusion injury. MBL-A was the main contributor to injury, likely owing to its earlier activation after ischemia and more efficient activation of the complement system than MBL-C.