We examined the role of tissue plasminogen activator‐ (tPA‐) matrix metalloproteinase‐ (MMP‐) 9 in mobilizing endothelial progenitor cells (EPCs) from bone marrow to circulation and critical limb ischemia (CLI) region. Male C57BL/6J mice having been irradiated were categorized into wild‐type mice (WT) receiving WT bone marrow cell (BMC) transfusion (group 1), WT mice receiving MMP‐9 knockout (MMP‐9−/−) BMC (group 2), MMP‐9−/− receiving MMP‐9−/− BMC (group 3), and MMP‐9−/− receiving WT BMC (group 4), each of which was subdivided into sham control (SC), CLI, SC‐tPA, and CLI‐tPA. In groups 1 and 4, by post‐CLI 18 h and day 14, circulating EPC (C‐kit+/CD31+, Sca‐1+/KDR+) levels were highest in CLI‐tPA subgroup. In groups 2 and 3, EPC levels did not differ among all subgroups. The EPC levels in bone marrow were higher in groups 2 and 3 than those in groups 1 and 4. By day 14, in animals with CLI, expression levels of proangiogenic factors (CXCR4, SDF‐1α, and VEGF) showed similar trends as circulating EPC levels. Moreover, the number of infiltrated neutrophils and macrophages in quadriceps was higher in groups 1 and 4 than groups in 2 and 3. In conclusion, tPA‐MMP‐9 axis plays a crucial role in EPC mobilization and angiogenesis in experimental CLI.