LIM kinase 1 (LIMK1) is a member of the LIMK family that has been considered to be involved in chemoresistance in various tumors, and N-methyladenosine (mA) is the most abundant nucleotide modification on mRNA. However, whether elevated expression of LIMK1 leads to chemoresistance due to mA modification remains to be further studied. The findings of our study indicate that high LIMK1 expression in colorectal cancer (CRC) cells promotes cell proliferation and increases resistance to 5-fluorouracil (5-FU). Moreover, downregulation of YTH domain-containing 2 (YTHDC2), an mA “reader”, in CRC cells resulted in decreased recognition and binding to the mA site “GGACA” in LIMK1 mRNA, thereby increasing LIMK1 mRNA stability and expression. Furthermore, the overexpression of LIMK1 facilitated eIF2α phosphorylation, which induced endoplasmic reticulum (ER) stress and promoted stress granule (SG) formation, ultimately leading to 5-FU resistance. This study evaluated the specificity of the YTHDC2/LIMK1/eIF2α signalling axis and the efficacy of related drugs in modulating 5-FU sensitivity in CRC.Copyright © 2023. Published by Elsevier B.V.