Cell migration plays an important role in many physiological and pathological processes, including tumor metastasis. Tumor cell migration is increased through the sequential induction of HIF-1α and VEGF under hypoxic conditions. Thymosin β-4 (Tβ4) is an actin-sequestering protein which controls cytoskeletal reorganization. Here, we investigated whether tumor cell migration could be co-operatively controlled by hypoxia and Tβ4. Cell migration was measured by wound healing assay with scratching confluent monolayers of tumor cells. Cell migration was enhanced 18 h after scratching cells. In addition, we found that the expression of HIF-1α, VEGF-A isoform 164/120 and Tβ4 was increased by scratching cells. Cell migration was decreased by the inhibition of Tβ4 or HIF-1α expression with lentiviral shRNA of Tβ4 or siRNA of HIF-1α, respectively. In contrast, cell migration was increased by the treatment with Tβ4 proteins. The inhibitory effect of Tβ4-shRNA or HIF-1α-siRNA was also attenuated by treatment with Tβ4 proteins. Collectively, these findings suggest that Tβ4 and HIF-1α cooperatively enhance tumor cell migration.