The aim of the present study was to investigate the role of cortactin in the infiltrative behavior of glioma cells and the potential mechanism of cortactin in promoting the migration and invasion of glioma cells. The expression of cortactin was detected by immunohistochemistry in 40 human glioma specimens and 8 non-tumor brain specimens. U251, LN229 and SNB19 glioma cells were employed for the in vitro study and assigned into the siRNA-cortactin (transfected with siRNA specific to cortactin), siRNA-NC (transfected with negative control RNA sequence) and siRNA-N (transfected with empty vector) groups. The expression of cortactin in different treated glioma cell groups was detected using western blot analysis and RT-qPCR. The migration and invasion of glioma cells under different treatments were assessed using a wound-healing assay and Transwell-chamber invasion assay, respectively. The lamellipodia of glioma cells following treatment were observed by immunofluorescence (IF) and changes of lamellipodia over time were imaged using an inverted microscope. The distribution of cortactin and the actin-related protein 2/3 (Arp2/3) complex in glioma cells were observed after IF detection. The expression of cortactin in the glioma specimens was significantly higher than that in non-tumor brain tissue (P<0.05) and positively correlated with the malignancy of glioma specimens (r=0.912, P=0.00). The cortactin expression in glioma cells was markedly inhibited (P<0.05) and their migration and invasion ability was also impaired significantly following treatment with siRNA (P<0.05) compared with the other two groups. The size and persistence time of lamellipodia were reduced after cortactin expression was inhibited in glioma cells. Cortactin and the Arp2/3 complex were co-localized in the front of glioma cells, where actin was polymerized and lamellipodia formed. Thus, the results revealed that, cortactin is crucial in invasion and migration of glioma cells, which may promote the migration and invasion of glioma cells by regulating lamellipodia formation, a process requiring the combination of cortactin and the Arp2/3 complex.