AbstractBackgroundN6‐methyladenosine (m6A) modification is an emerging epigenetic regulatory mechanism in tumourigenesis. Considering that AlkB homolog 5 (ALKBH5) is a well‐described m6A demethylase in previous enzyme assays, we aimed to investigate the role of m6A methylation alteration conferred by disturbed ALKBH5 in colorectal cancer (CRC) development.MethodsExpression of ALKBH5 and its correlation with clinicopathological characteristics of CRC were evaluated using the prospectively maintained institutional database. The molecular role and underlying mechanism of ALKBH5 in CRC were explored using in vitro and in vivo experiments with methylated RNA immunoprecipitation sequencing (MeRIP‐seq), RNA‐seq, MeRIP‐qPCR, RIP‐qPCR and luciferase reporter assays.ResultsALKBH5 expression was significantly upregulated in CRC tissues compared to the paired adjacent normal tissues, and higher expression of ALKBH5 was independently associated with worse overall survival in CRC patients. Functionally, ALKBH5 promoted the proliferative, migrative and invasive abilities of CRC cells in vitro and enhanced subcutaneous tumour growth in vivo. Mechanistically, RAB5A was identified as the downstream target of ALKBH5 in CRC development, and ALKBH5 posttranscriptionally activated RAB5A by m6A demethylation, which impeded the YTHDF2‐mediated degradation of RAB5A mRNA. In addition, we demonstrated that dysregulation of the ALKBH5‐RAB5A axis could affect the tumourigenicity of CRC.ConclusionsALKBH5 facilitates the progression of CRC by augmenting the expression of RAB5A via an m6A‐YTHDF2‐dependent manner. Our findings suggested that ALKBH5‐RAB5A axis might serve as valuable biomarkers and effective therapeutic targets for CRC.