Background <p>Dysregulation of N6-methyladenosine (m6A) has been implicated in the pathophysiology of various autoimmune diseases. However, its role in dermatomyositis (DM), particularly in cases associated with anti-MDA5 antibodies, remains unclear. This study aimed to elucidate the potential involvement of m6A modifications of mRNAs in the pathogenesis of anti-MDA5⁺DM.</p> Methods <p>We assessed mRNA m6A methylation levels in peripheral blood mononuclear cells (PBMCs) from anti-MDA5⁺DM patients and healthy controls (HC) using LC-MS/MS assay. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western blotting were conducted to determine the mRNA and protein expression levels of YTHDF2 in PBMCs and monocytes from anti-MDA5⁺DM patients and HC. RNA sequencing (RNA-seq) was performed to identify differentially expressed genes and signaling pathways in siYTHDF2-THP-1 cells. RNA immunoprecipitation and quantitative PCR (RIP-qPCR) were used to explore the interaction between YTHDF2 protein and IFNB mRNA in THP-1 cells.</p> Results <p>The overall level of mRNA m6A methylation was found to be decreased in PBMCs of anti-MDA5⁺ DM compared to HC. The expression levels of YTHDF2 were downregulated in PBMCs and monocytes of anti-MDA5⁺DM patients compared with HC. The expression of IFNB was increased in PBMCs and monocytes of anti-MDA5⁺DM. Knockdown of YTHDF2 in THP-1 cells significantly increased IFNB expression and activated the JAK-STAT signaling pathway. The interaction between YTHDF2 protein and IFNB mRNA was confirmed by RIP-qPCR. The upregulated expression of type I IFN caused by YTHDF2 knockdown in THP-1 cells could be inhibited by JAK inhibitors.</p> Conclusions <p>Our findings suggest a decrease in YTHDF2 expression in anti-MDA5⁺DM monocytes, potentially enhancing IFNB expression and promoting the activation of JAK-STAT signaling pathway.</p>