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Cancer stemness is a major therapeutic challenge in oncology. This study investigated the functional role and molecular mechanism of cell division cycle-associated 8 (CDCA8) in non-small cell lung cancer (NSCLC) stem cells. In this study, NSCLC and paracancerous tissues were collected. The lung adenocarcinoma cell line A549 and the lung squamous cell carcinoma cell line NCI-H520 were used. The stem-like cell population in A549 and NCI-H520 was isolated by CD44+ fluorescence-activated cell sorting. Gene expression was detected by quantitative real-time PCR, western blotting, and immunohistochemical staining. Cell stemness was assessed by biomarker (SOX and NANOG) expression detection, colony formation assay, and sphere-formation assay. Cell migration and invasion ability were determined by the Transwell experiment. Our results showed that CDCA8 expression was higher in NSCLC tissues than in paracancerous tissues. CDCA8 overexpression enhanced stemness properties, as evidenced by increased biomarker expression and colony formation, larger sphere size, and enhanced migratory/invasive capacity. Conversely, CDCA8 knockdown had the opposite effect. Mechanistically, we identified Y-box binding protein 1 (YBX1) as a direct binding protein of CDCA8 mRNA that positively regulated CDCA8 expression. YBX1 overexpression had a similar effect to CDCA8. Furthermore, recovery experiments revealed that the stemness-promoting effect of YBX1 was reversed by CDCA8 knockdown. These findings were further validated in xenograft models, confirming that the YBX1/CDCA8 axis promoted tumorigenesis in vivo. Collectively, our study reveals that YBX1 enhances cell stemness and metastasis of NSCLC by promoting CDCA8 expression. Our findings established a new mechanism that maintains NSCLC stemness and may provide novel biomarkers.