Abstract: Background　Anti-MDA5 (Melanoma differentiation-associated gene 5) antibody-positive dermatomyositis (DM) is frequently complicated by interstitial lung disease (ILD), particularly the rapidly progressive ILD (RPILD), which confers a poor prognosis. The precise mechanisms driving the aberrant activation of the MDA5 pathway in this condition remain unclear. Objective　To investigate whether viral infection is a driving factor in anti-MDA5 antibody-positive DM with ILD, and to elucidate its role in mediating MDA5 pathway hyperactivation through transcriptional and post-translational mechanisms. Methods　Ant-MDA5 antibody-positive DM patients and anti-synthetase syndrome (ASS) patients diagnosed at the First Medical Center of PLA General Hospital between December 2023 and January 2025 were enrolled. Comparative analysis of clinical characteristics and immunological parameters was performed between the two groups. Bronchoalveolar lavage fluid (BALF) was collected from 15 anti MDA5 antibody-positive DM-ILD patients for metagenomic next-generation sequencing (mNGS) to analyze the pathogen landscape. For in vitro experiments, THP-1 cells were infected with human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), influenza A virus (IAV), or human coronavirus OC43 (HCoV-OC43) to establish infection models. The expression levels of MDA5 and its downstream signaling molecules were detected using reverse transcription quantitative real-time PCR (RT-qPCR) and Western blotting. The effect of viral infection on MDA5 protein stability was assessed via a cycloheximide (CHX) chase assay. Results　This study enrolled a total of 20 anti-MDA5 antibody-positive DM patients and 10 ASS patients. Compared with the ASS group, the anti-MDA5 antibody-positive DM group had a significantly higher incidence of skin ulcers (45.0% vs 0%, P＜0.05) and RPILD (55.0% vs 0%, P＜0.01). Additionally, peripheral blood CD4⁺ T cell counts (345.9±228.9 μL vs 757.6±526.7 μL) and CD8⁺ T cell counts (154.2±119.1 μL vs 391.1±247.3 μL) were significantly lower, while serum IL-6 levels (14.2±10.8 pg/mL vs 4.3±4.4 pg/mL) were significantly higher (all P＜0.05). Metagenomic sequencing of BALF from 15 anti-MDA5 antibody-positive DM-ILD patients revealed detectable viral infections in 66.7% (10/15), with HCMV and HSV-1 detection rates of 46.7% (7/15) and 26.7% (4/ 15), respectively. In vitro experiments demonstrated that HCMV and HSV-1 infection promoted the expression of MDA5 and its downstream signaling molecules. In contrast, IAV and HCoV-OC43 infection enhanced MDA5 protein stability by prolonging its half-life without significantly altering its mRNA levels. Conclusion　Viral infection may contribute to the pathogenesis of anti MDA5 antibody-positive DM complicated by ILD via a dual mechanism: inducing transcriptional activation of MDA5 and enhancing its protein stability, leading to sustained activation of the MDA5 signaling pathway.