Background: Acute exacerbation is regarded as a risk factor for prediction of poor outcome in patients with interstitial lung disease. There is no unified view on the cause of acute exacerbation of interstitial lung disease (AE-ILD), among which infection is considered as an important cause, however, there are few related studies. Metagenomic next-generation sequencing (mNGS) has been applied for pathogen determination for infectious diseases. However, the clinical significance of mNGS in AE-ILD remains unclear.Methods: We retrospectively reviewed 33 cases confirmed of AE-ILD from Respiratory and Critical Care Medicine at Affiliated Hospital of Xuzhou Medical University in Jiangsu, China, between Jun 2020 and May 2021. Patients were enrolled and the potential pathogens in bronchoalveolar lavage fluid (BALF) or deep sputum were detected simultaneously by conventional microbial examination and mNGS. We evaluated the etiological diagnostic effect of mNGS in patients with AE-ILD.Results: In 96.8% (32/33) of the patients with AE-ILD, various pathogens were determined positively by mNGS, including 46% infected with viral, 28% with bacterial and 26% with fungal. Of these, 78% were considered to have mixed infection, mainly manifested as fungus + virus (39.74%), fungus + virus + bacteria (32.05%), and bacteria + virus (28.21%). In addition, the overall survival after admission for AE-ILD was 42.42% at 1 month and 51.51% at 3 months. Conclusions: mNGS showed that infection played an important role during the acute exacerbation of ILD, there are a variety of pathogens including bacteria, viruses and fungi, among which mixed infections were common.