Abstract Thymosin β4 (Tβ4) is a developmentally expressed 43-amino acid peptide that inhibits organization of the actin-cytoskeleton by sequestration of G-actin monomers. Tβ4 improves cardiac function after myocardial infarction in adult mice and promotes healing properties in both dermal and corneal wounds. We tested the hypothesis that Tβ4 improves functional neurological outcome in a rat model of embolic stroke. Experimental Procedures: Male Wistar rats ( n=18) were subjected to embolic middle cerebral artery occlusion (MCAo). Tβ4 (6 mg/kg, IP) was administered 24 h after MCAo and then every 3 days for four additional doses ( n=9). Rats treated with saline were used as a control ( n=9). The adhesive-removal test (ART) and modified Neurological Severity Score (mNSS) were performed to measure functional outcome. Rats were sacrificed 56 days after MCAo. Immunostaining was performed with antibodies against NG-2 (chondroitin sulfate proteoglycan), CNPase (2”, 3”-cyclic nucleotide 3'-phosphodiesterase) to detect immature and mature oligodendrocytes. Neurofilament-H (NF-H) antibodies were used to detect axons while myelinated axons were identified with Bielschowsky/Luxol (B/L) Blue staining. EBA (endothelial barrier antigen) was used for detection of mature vessels. Results: Ischemic rats treated with Tβ4 demonstrated a significant overall improvement ( P<0.01) in the ART and the mNSS when compared to controls. Significant improvement was observed beginning at 14 and 35 days, respectively. Lesion volumes showed no significant differences between the two groups. Treatment with Tβ4 increased myelinated axons and increased vessel density in the ischemic boundary ( P<0.05) and augmented remyelination which was associated with an increase of oligodendrocyte progenitor cells (OPCs) and myelinating oligodendrocytes ( P<0.05). Conclusions: The present study suggests that Tβ4 improves neurological functional outcome after embolic stroke in rats. Axonal remodeling from mobilization of OPCs is proposed as contributing to Tβ4 induced functional improvement.