Loss of monoubiquitination of histone H2B (H2Bub1) was found to be associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma. This study investigated the association and impact of the ubiquitin-specific peptidase 22 (USP22), an H2Bub1 deubiquitinase, on stem cell-like characteristics and cisplatin resistance in cancer-initiating cells (CIC) from primary lung adenocarcinoma. CICs were isolated, enriched, and characterized from patient-derived cancer tissues using both in vitro tumorsphere formation and in vivo xenograft assays. USP22 was determined to be predominantly expressed in CICs, a subpopulation of cells with high expression of the stem cell biomarkers, CD133 and CD44. The expression of USP22 in CICs is markedly reduced upon FBS/retinoic acid-induced differentiation. Moreover, knockdown of USP22 significantly suppressed tumorsphere formation and xenograft growth in NOD-SCID gamma (NSG) mice. Notably, USP22 and aldehyde dehydrogenase (ALDH) activity were elevated in tumorsphere cells that survived cisplatin treatment, whereas knockdown of USP22 significantly sensitizes tumorsphere cells to cisplatin. Interestingly, ALDH1A3, a predominant ALDH isozyme implicated in enhancing cisplatin resistance in lung adenocarcinoma, is significantly downregulated upon knockdown of USP22 in tumorsphere cells. Furthermore, knockdown of ALDH1A3 significantly sensitizes tumorsphere cells to cisplatin. Combined, these data demonstrate that USP22, predominantly expressed in CD133 + CICs, plays a critical role in tumorigenicity and cisplatin resistance in lung adenocarcinoma. Implications: Targeting USP22 represents a potential therapeutic approach to suppress CICs in lung adenocarcinoma partially through downregulation of ALDH1A3 expression. Mol Cancer Res; 16(7); 1161–71. ©2018 AACR .