The enzymatic hydrolysis of the extract ofSophora japonicaby two glycosyl hydrolases (hesperidinase and galactosidase) was performed in order to obtain kaempferol (KPF)-enriched extract with an enhanced anticancer activity. The current study examined the effectiveness of bothSophora japonicaextracts (before (KPF-BBR) and after (KPF-ABR) bioconversion reactions) in reducing cell viability and inducing apoptosis in human high-degree gliomas in vitro. Cytotoxicity was determined using an MTT assay. The effects of both compounds on the proliferation of glioma cell lines were measured using trypan blue exclusion, flow cytometry for cell cycle, wound healing (WH), and neurosphere formation assays. Cellular apoptosis was detected by DNA fragmentation and phosphatidylserine exposure. qPCR and luciferase assays evaluated NF-kB pathway inhibition. The survival rate of NG-97 and U-251 cells significantly decreased in a time- and dose-dependent manner after the addition of KPF-BBR or KPF-ABR. Thus, a 50% reduction was observed in NG-97 cells at 800 µM (KPF-BBR) and 600 µM (KPF-ABR) after 72 h. Both compounds presented an IC50 of 1800 µM for U251 after 72 h. The above IC50 values were used in all of the following analyses. Neither of the KPF presented significant inhibitory effects on the non-tumoral cells (HDFa). However, after 24 h, both extracts (KPF-BBR and KPF-ABR) significantly inhibited the migration and proliferation of NG-97 and U-251 cells. In addition,MMP-9was downregulated in glioma cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) plus KPF-BBR and TPA+KPF-ABR compared with the TPA-treated cells. Both KPF-BBR and KPF-ABR significantly inhibited the proliferation of glioma stem cells (neurospheres) after 24 h. DNA fragmentation assays demonstrated that the apoptotic ratio of KPF-ABR-treated cell lines was significantly higher than in the control groups, especially NG-97, which is not TMZ resistant. In fact, the flow cytometric analysis indicated that KPF-BBR and KPF-ABR induced significant apoptosis in both glioma cells. In addition, both KPF induced S and G2/M cell cycle arrest in the U251 cells. The qPCR and luciferase assays showed that both KPFs downregulatedTRAF6,IRAK2, IL-1β,andTNF-α, indicating an inhibitory effect on the NF-kB pathway. Our findings suggest that both KPF-BBR and KPF-ABR can confer anti-tumoral effects on human cell glioma cells by inhibiting proliferation and inducing apoptosis, which is related to the NF-κB-mediated pathway. The KPF-enriched extract (KPF-ABR) showed an increased inhibitory effect on the cell migration and invasion, characterizing it as the best antitumor candidate.