Temozolomide (TMZ) is currently the first-line drug used for clinical postoperative or non-surgical chemotherapy for glioma, but acquired and intrinsic resistance to TMZ limits its application. The anti-proliferative effect of formononetin on human glioma cells had been confirmed. To improve therapeutic effects of TMZ, we studied the effect of formononetin in combination with TMZ on C6 glioma cells. The anti-proliferative effect of C6 cells was tested by 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. The synergy was evaluated by Chou–Talalay method. Morphological changes were observed by hematoxylin–eosin (HE) staining. The effect of formononetin in combination with TMZ on apoptosis of C6 cells was investigated by flow cytometry. The effect of formononetin in combination with TMZ on migration of cells was investigated by wound healing assay and transwell assay. The expression of proteins related to apoptosis and migration were detected by Western blot. These results showed that formononetin or TMZ alone could inhibit the growth of C6 cells in dose-dependent manner and formononetin in combination with TMZ had synergy effect on C6 cells. Further changes in cell morphology could be observed in drug combination by HE staining. Drug combination enhanced the expression of Bax, Cleaved Caspase-3, Cleaved Caspase-9, decreased the expression of Bcl-2, and promoted tumor cells apoptosis. In addition, the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were down-regulated via drug combination which resulted into inhibiting migration of C6 cell. In conclusion, formononetin in combination with TMZ can play a synergistic role in anti-C6 cells, the mechanisms of synergy depended on multiple pathways.