Background <p>Ubiquitin-conjugating enzyme E2S (UBE2S) has been implicated in the development of various types of cancer. We previously showed that UBE2S is highly expressed and plays an oncogenic role in lung adenocarcinoma (LUAD). The aim of this study was to elucidate the molecular mechanisms underlying the function of UBE2S in the progression of LUAD.</p> Methods <p>The interaction between UBE2S and its binding proteins was explored using immunoprecipitation coupled with mass spectrometry and coimmunoprecipitation assays. The expression of UBE2S in clinical tissues and LUAD cells was detected by western blotting. The downstream targets of UBE2S identified in our previous microarray study were analyzed and verified using a luciferase assay and chromatin immunoprecipitation–quantitative polymerase chain reaction. In vitro and in vivo functional studies were conducted to confirm the mechanisms involving UBE2S in the progression of LUAD.</p> Results <p>An interaction between UBE2S and IκBα was detected, and UBE2S was negatively correlated with IκBα and positively correlated with p65 in clinical samples of LUAD. Glycine amidinotransferase (GATM) was identified as a downstream gene of UBE2S in LUAD cells. The expression of GATM was negatively regulated by UBE2S and restored by p65 expression. Moreover, we confirmed the binding of p65 to the GATM promoter. GATM was more highly expressed in LUAD tissues than in paracarcinoma tissues. A functional study verified the oncogenic role of UBE2S in the progression of LUAD through GATM/creatine activity. Furthermore, animal experiments confirmed the role of UBE2S in promoting the growth of LUAD tumors through the p65/GATM axis in vivo.</p> Conclusions <p>The newly identified UBE2S/NF-κB/GATM axis may be a target for the treatment of LUAD.</p>