Objective: To examine the expression of ZBTB3 in human glioblastoma (GBM) tissues and cell lines, and to explore the effects of ZBTB3 on the proliferation and clonal formation of GBM cells and their regulatory mechanism. Methods: The expression of ZBTB3 in tumor tissues of GBM patients was analyzed by GEPIA2 database. The mRNA and protein expression levels of ZBTB3 in GBM cell lines (U251, U373, U87) were detected by RT-PCR and Western blot, and U87 cell line was identified with the highest expression of ZBTB3. CCK-8 and clonal formation assay were used to examine the effects of silencing ZBTB3 on the proliferation and clonal formation of U87 cells. U87 cells were treated with p38 MAPK, AMPK and Akt1 inhibitors, and the phosphorylation levels of p38 MAPK, AMPK and Akt1 were detected by Western blot, ZBTB3 mRNA and protein levels were detected by RT-PCR and Western blot, cell proliferation and clone formation were examined by CCK-8 and clone formation. Results: The expression of ZBTB3 in tumor tissues of GBM patients was significantly higher than that in normal tissues. The expression levels of ZBTB3 in U251, U373 and U87 cell lines were examined, and the highest expression in U87 cells was observed. Silencing ZBTB3 markedly inhibited the proliferation and clonal formation of U87 cells. AMPK inhibition could not only obviously reduce the expression level of ZBTB3 in U87 cells, but also markedly attenuate the proliferation and clonal formation of U87 cells. Conclusion: The expression of ZBTB3 is obviously increased in GBM tissues and cells, and the AMPK-up-regulated ZBTB3 expression promotes the proliferation and clonal formation of GBM cells.