Objective:To explore the molecular mechanism that palmitoyl acyltransferase 20(ZDHHC20)participates in the pathogenesis of colorectal cancer(CRC)through Palmitoylation modification to regulate 5-methylcytosine(m5C)reader Y-box binding proteinl(YBX1)protein stability,and improve mRNA m5C modification level.Methods:The expression of ZDHHC20 mRNA and pro-tein in CRC was analyzed by bioinformatics.Further analysis of the association among ZDHHC20 expression levels and the clinicopatho-logical features of CRC patients was performed.The expression level of ZDHHC20 in CRC cells was verified using RT-qPCR.A CCK-8 assay was used to detect the effect of ZDHHC20 on the proliferation of CRC cells.Transwell was used for cell invasion assays.The rela-tionship between the expression of ZDHHC20 and YBX1 in CRC and the palmitoylation site of YBX1 was investigated by bioinformatics database analysis.Palmitoylation protein was enriched by ABE method and Westernblot experiment was used to detect YBX1 palmitoyla-tion modification in CRC.Overexpression of ZDHHC20 was used to detect the palmitoylation modification level of YBX1.The Western blot was used to measure the stability of YBX1 protein under 2-BP and actinomycin.The influence of YBX1 on the proliferation and mi-gration of CRC cells was measured by CCK-8 and Transwell.LC-MS was used to analyze the impact of YBX1 on m5C modification level in CRC cells.Results:An upregulation of ZDHHC20 mRNA and protein were observed in the sample CRC cells and tissues(P＜0.05).ZDHHC20 expression was correlated with age,tumor stage and lymph node metastasis(P＜0.05).Overexpression of ZDHHC20 contributes to proliferation and invasion in CRC cells(P＜0.05).The expression between ZDHHC20 and YBX1 were posi-tively correlated(P＜0.05),and YBX1 had multiple palmitoylation modification site.YBX1 was a palmitoylated protein and ZDHHC20 could increase YBX1 palmitoylation modification level and improve the stability of YBX1 protein(P＜0.05).YBX1 could enhance CRC cell proliferation and invasion.Also YBX1 could improve m5C modification level in CRC(P＜0.05).Conclusion:ZDHHC20 can improve the stability of YBX1 protein through Palmitoylation modification and mRNA m5C modification level,and enhance CRC cell proliferation and invasion.