Abstract Background: The prognosis of recurrent and metastatic Ewing sarcoma (EWS) is extremely poor and the problem of drug resistance is significant; thus, it is extremely important to identify the mechanisms underlying chemotherapy resistance. This study aimed to explore the genes associated with chemotherapy resistance in EWS. Procedure: Gene expression profile data of GSE12102 and GSE63155were downloaded from GEO to screen for differentially expressed genes (DEGs). GO functional annotation analysis was performed on common DEGs. Twenty-one patients were divided into good response (tumor necrosis rate ≥90%) (n=4) and poor response (tumor necrosis rate <90%) (n=17) groups to chemotherapy. Formalin-fixed paraffin-embedded (FFPE) tissues of 21 patients after chemotherapy and 5 patients who are in poor response group before chemotherapy were collected. Real-Time Quantitative Reverse Transcription PCR(qRT-PCR) was used to verify gene expression differences.. The relationship between prognosis and gene expression level verified by GSE17679 Results: The PGAP6 expression level after chemotherapy in the poor response group (2.78 ± 2.36) was higher than that in the good response group (1.16 ± 0.65) (P = 0.024). Expression of PGAP6 after chemotherapy (16.70 ± 13.78) was higher than that before chemotherapy (1.16 ± 0.62; P = 0.071). Verified by external datasets, the expression level of PGAP6 was related to prognosis. Conclusions: PGAP6 drives EWS chemoresistance and may be associated with acquired resistance. The expression levels of PGAP6 correlated with EWS prognosis. PGAP6 can be used as a new target for reversing chemotherapy resistance of EWS. KEYWORDS: Ewing sarcoma; chemotherapy; drug resistance; PGAP6; TMEM8A