OBJECTIVE Although many efforts on revealing mechanism of the constitutive activation of NF-κB in cancer cells contributed to understanding canonical pathways, largely it remains to be determined for therapeutic approaches. Recently, we found that increased expression of transglutaminase 2 (TGase 2) appears to be responsible for constitutive activation of NF-κB in certain types of cancer cells. In previous studies, we demonstrated that TGase 2 inhibition markedly increases anti-cancer drug sensitivity in drug resistance cancer cells. Therefore, we develop safe and effective TGase 2 inhibitors for therapeutic approach. METHODS We screened a chemical library of natural compounds using in vitro TGase 2 activity assay. The salient discovery was that glucosamine (GlcN), a known anti-inflammatory substance, inhibited the cross-linking activity of TGase 2. We tested, through a biochemical analysis including kinetics, whether the GlcN and GlcN analogs specifically inhibit TGase 2. We also determined the inhibitory mechanism using conformational change of TGase 2. RESULTS We found that the primary amine of GlcN plays a key role in TGase 2 inhibition. We also demonstrated that GlcN reversed TGase 2-mediated I-κBα polymerization in vitro. Interestingly, the metabolite of GlcN, glucosamine-6-phosphate (GlcN6P), inhibited TGase 2 activity via binding to the GTP-binding site with better efficiency than GlcN. In the native gel electrophoresis, it was clearly observed that GlcN6P binds to TGase 2 directly as an allosteric inhibitor. CONCLUSIONS We concluded that GlcN inhibits TGase 2 activity by direct contact. GlcN and its metabolite GlcN6P can down-regulate constitutive activation of NF-κB in vivo via inhibition of TGase 2.