Abstract: Objective To explore the clinical characteristics of severe pneumonia in children caused by human adenovirus type 7 (HAdV7) and the application of metagenomic next-generation sequencing (mNGS) in etiological diagnosis. Methods The patients with severe pneumonia associated with HAdV7 infection were enrolled from Pediatric Department of Anhui Provincial Hospital Affiliated to Anhui Medical University from January 2019 to November 2019. HAdV7 infection was identified by mNGS of blood or alveolar lavage fluid. The clinical data and results of mNGS of included patients were retrospectively analyzed. Results Twenty patients were included, and 17 of them were males. Twelve cases (60%) were aged from 6 months to 2 years, which was the most common age group. A total of 19 cases had high fever (>39 ℃). Fever duration of 16 cases was more than 10 days. There were 15 cases with both of cough and wheezing. Complications included toxic encephalopathy in 1 case, heart failure in 7 cases, respiratory failure in 5 cases and pleural effusion in 5 cases. On admission, WBC was (8.6±4.3)×109·L-1, and there were 6 cases with more than 10×109·L-1 and 2 cases with less than 4×109·L-1. The percentage of neutrophils was (55.9±131)%. The median of C-reactive protein was 10.9 (5.6, 23.7) mg·L-1, and there were 2 cases with more than 30 mg·L-1. As the disease aggravated such as hypoxemia or dyspnea, the median of WBC was 8.3 (3.1, 17.6) ×109·L-1, and there were 8 cases with more than 10×109·L-1 and 6 cases with less than 4×109·L-1; the percentage of neutrophils became (65.4±17.2)%, and there was an increase in 16 cases; the median of C-reactive protein became 26.5 (12.2, 65.9) mg·L-1, and there were 8 cases with more than 30 mg·L-1. Chest CT showed consolidation and air bronchogram in bilateral lungs were found in 17 cases respectively. Nineteen cases improved and 1 case died. The median of identified HAdV7 DNA sequences was 1,671.0 (892.0, 2 467.8) and the coverage rates were 98.9(89.8, 99.1)%. Of the 20 cases, 10 were performed mNGS of blood and 10 were performed mNGS of alveolar lavage fluid. The median of the identified reads of the two groups were 1,152.0 (347.8, 1,991.5) and 2,033.5 (1,263.2，2,901.8), and the coverage rates were 98.9(81.6, 99.1)% and 99.0(89.9, 99.2)%, respectively. Thirteen cases were affected by HAdV7 infection alone, and 7 cases were affected by mixed infection. Conclusion mNGS of blood or alveolar lavage fluid could identify HAdV7 infection accurately. Doctors should be aware of severe pneumonia and complications caused by HAdV7 infection and perform mNGS as soon as possible, when infants younger than 2 years old had persistent high fever, cough, wheezing, increased or decreased WBC focusing on neutrophils, high C-reactive protein and consolidation and air bronchogram in bilateral lungs shown by chest CT.