AbstractBackgroundNOP2/Sun domain 2 (NSUN2) is one of the important RNA methyltransferases catalyzing 5‐methylcytosine (m5C) formation and participates in many critical bioprocesses. However, the roles and underlying molecular mechanisms of NSUN2‐mediated m5C modification in colorectal cancer (CRC) remain unclear.MethodsTo explore the NSUN2 expression in CRC, fresh tissue samples were collected and Nsun2 knockout mouse was constructed. In vitro and in vivo functional assays were conducted to assess the role of NSUN2. RNA array and bisulfite sequencing were used to investigate the potential targets. The mechanisms of NSUN2 function on SKIL were identified by m5C‐methylated‐RNA immunoprecipitation and RNA stability assays. Additionally, tissue microarray analysis was conducted and patient‐derived tumour xenograft mouse (PDX) models were used to define the potential therapeutic targets.ResultsNSUN2 was highly expressed in CRC and correlated with poor CRC patient survival. Moreover, silencing NSUN2 suppressed CRC tumourigenesis and progression in Nsun2 knockout mouse models. In vitro and in vivo studies suggested that NSUN2 promoted colorectal cancer cell growth. Mechanistically, SKI‐like proto‐oncogene (SKIL) is positively regulated by NSUN2, and the NSUN2‐SKIL axis is clinically relevant to CRC. NSUN2 induced m5C modification of SKIL and stabilized its mRNA, which was mediated by Y‐box binding protein 1 (YBX1). Elevated SKIL levels increased transcriptional coactivator with PDZ‐binding motif (TAZ) activation.ConclusionsOur findings highlight the importance of NSUN2 in the initiation and progression of CRC via m5C‐YBX1‐dependent stabilization of the SKIL transcript, providing a promising targeted therapeutic strategy for CRC.