Glioma, the most frequent central nervous system malignancy, is often promoted by the overexpression of Fos-like antigen 1 (FOSL1). However, the regulation of FOSL1 remains unexplored. The present study aimed to investigate the regulatory mechanism of FOSL1 to identify potential therapeutic targets for glioblastoma. This study's initial investigation utilized dual-luciferase reporter gene assays and quantitative polymerase chain reaction (qPCR) assays to establish that Kruppel-like factor 14 (KLF14) inhibits the transcription of FOSL1. Subsequent immunohistochemistry and western blotting (WB) assays on glioma tissues confirmed a negative association between FOSL1 and KLF14. This study generated KLF14 knockdown cells and double knockdown cells of KLF14 and FOSL1 and further assessed cell growth through various experimental methods. The impact of KLF14 on tumor cell migration via FOSL1 was determined using qPCR and WB assays. A xenograft tumor model was utilized to verify tumor growth suppression by KLF14. The present study demonstrated that KLF14 restrains FOSL1 transcription and is inversely correlated with FOSL1 in glioma tissues. KLF14 overexpression was found to counteract FOSL1's effect on cell migration and epithelial-to-mesenchymal transition in glioma cells, which coincided with decreased Snail2 and cluster of differentiation 44 (CD44) expressions. Further, KLF14 overexpression was shown to hinder tumor progression in vivo. This study highlights that FOSL1 is negatively regulated by KLF14 in glioblastoma and suggests that KLF14 overexpression can mitigate tumor growth by inhibiting FOSL1, thus identifying KLF14 as a novel molecular target for treating glioblastoma. Further research into the interplay and regulatory dynamics between KLF14 and FOSL1 under varying stress conditions can enhance the precision of glioblastoma treatment.