Background <p>Laminin subunit beta 1 (LAMB1) has regulatory functions on the proliferation, attachment, and migration of tumor cells, with increased levels linked to different cancers.</p> Objective <p>This study aims at investigating the effects and mechanisms of LAMB1 in glioma.</p> Methods and material <p>Glioma cell models with LAMB1 overexpression or downregulation were constructed. Cell viability, proliferation, and invasion were evaluated. Glucose uptake and lactate production were examined, and Seahorse was used to assess the extracellular acidification rate (ECAR). The EC50 of temozolomide (TMZ) in glioma cells was tested. Western blotting was conducted to monitor the expression of HK1, HK2, PDHA, and PKM. Bioinformatic analysis was employed to investigate the downstream mechanism of LAMB1. In addition, a subcutaneous tumor model was constructed to determine the influence of LAMB1 on GBM cell growth in vivo.</p> Results <p>LAMB1 overexpression enhanced cell viability, proliferation, and invasion and promoted glioma cell growth. LAMB1 upregulation enhanced cellular glycolysis and repressed the sensitivity of cells to TMZ. LAMB1 activated the NF-κB pathway. Downregulation of LAMB1 or mitigating of the NF-κB pathway by Bay 11-7082 inhibited glioma cell proliferation, growth, and glycolysis and enhanced TMZ sensitivity.</p> Conclusions <p>LAMB1 downregulation exerted antitumor effects on glioma cells by regulating the NF-κB/HK2 axis.</p>