Cell migration is an important factor influencing the treatment outcomes of high-grade glioma (World Health Organization grades III-IV). Using immunohistochemical staining, the present study demonstrated that the protein levels of phosphorylated pyruvate dehydrogenase α1 (p-PDHA1) were increased according to the grade of glioma. Moreover, p-PDHA1 mediated tumor necrosis factor-α (TNF-α)-induced cell migration in glioma cells. Phalloidin staining and western blot analysis were used to detect the protein level of p-PDHA1 in U251 glioma cells stimulated by TNF-α at different time points. Phalloidin staining was used to observe the cytoskeletal structure. The effects on the expression of specific migration markers and on the cytoskeletal structure were also detected. Dichloroacetic acid is an inhibitor of PDK. These results indicated that p-PDHA1 served an important role in the migration of glioma cells, and consequently in the development of glioma.