Recombinant thymosin β4 (rTβ4) has been reported to migrate and promote vascularization, wound-healing, and hair growth in a mouse hindlimb ischemia model of peripheral vascular disease. C57BL/6 mice (11-weeks-old) were anesthetized and an ischemic model was made by cutting the right aorta femoralis. The ischemic group was intraperitoneally administered with saline (300 μL/mouse) and the muscular administration group received rTβ4 (150 μg in 300 μL of saline) or rTβ4 (150 μg in 300 μL saline) to the abdominal cavity at 3-day intervals for 21 days. Myoatrophy of the ischemic group was observed compared to the normal control group. Generation of adjacent vessels was carried out in the rTβ4 administration group compared to the ischemic group. The biopsy results showed significant fibrosis around the muscular undersurface and perimysium in the musculus quadriceps femoris of the ischemic group, whereas partial fibrosis was observed in the perimysium and endomysium in the rTβ4 administration group. Immunostaining indicated that expression levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor-1 (VEGF-1), and endothelial nitric oxide synthase (eNOS) in the rTβ4 group were higher than those of the ischemic group. Western blotting showed that expression levels of HIF-1α, VEGF-1, and eNOS in the rTβ4 group were higher than those of the ischemic group. In conclusion, rTβ4 increases expression levels of HIF-1α, VEGF-1, and eNOS, resulting in angiogenesis.