Cancer stem-like cells (CSCs) are regarded as sources of tumorigenesis, metastasis, and drug resistance, which limits current cancer therapies. Elucidating the molecular modes governing CSC properties is necessary to optimize therapeutic approaches. In this study, we discovered that ubiquitin-protein ligase E3C (UBE3C)-mediated ubiquitination is a key posttranslational mechanism involved in maintaining CSC properties of non-small-cell lung cancer (NSCLC). UBE3C was overexpressed in stem-like NSCLC cells and acted as a stemness enhancer. Knockdown of UBE3C reduced NSCLC stemness and tumorigenesis both in vivo and in vitro. We further identified AHNAK as a novel UBE3C substrate, finding that UBE3C maintained stemness by ubiquitinating and promoting AHNAK degradation. AHNAK functioned as a cofactor assisting p53 binding to stemness-related gene promoters to inhibit transcription. Subsequent downregulation of AHNAK by UBE3C overexpression removed p53-mediated inhibition of gene expression, resulting in enhanced stemness. Clinical significance was investigated in 208 NSCLC patients and confirmed that attenuated UBE3C activity and elevated AHNAK protein levels correlated with extended survival time. Collectively, findings reveal the first global characterization of UBE3C-mediated ubiquitination as a key regulator of CSCs, with results suggesting involvement of the AHNAK-p53 complex.