Objective To understand the epidemiological and clinical characteristics of adenovirus healthcare-associated infection (HAI) in children, and provide basis for targeted prevention and treatment. Methods Medical data of children admitted to a children's hospital from January 2011 to December 2020 due to adenovirus infection were analyzed retrospectively. Children detected adenovirus in respiratory secretion over 48 hours and less than 48 hours after admission were divided into HAI group and community-associated infection group (CAI group) respectively. Epidemiological and clinical characteristics of two groups of children were compared. Results A total of 1 158 children with adenovirus pneumonia received medical treatment, including 55 (4.75%) HAI cases and 1 103 (95.25%) CAI cases. The average age of 55 children in HAI group was (2.83±0.38) years old, and the main onset age was less than 3 years old (n=36, 65.45%). In HAI group, 49.09% children had severe pneumonia, higher than 6.98% in CAI group, with statistically significant difference (P < 0.05). The case fatality rate of children in HAI group and CAI group were 1.82% and 0.18% respectively. 94.55% children in HAI group had fever. Fever duration was (10.80±0.93) days, higher than that ([6.06±0.13] days) in CAI group, with statistical difference (P < 0.001). In HAI group, 32.73% children had underlying diseases. The percentages of moderate and severe anemia (10.91%), congenital heart disease (7.27%), blood and tumor diseases (5.45%), and nervous system diseases (7.27%) were higher than that of CAI group (1.63%, 1.36%, 0.36%, and 1.09% respectively), all were statistically different (all P < 0.05). Primary diseases in children in HAI group were mainly respiratory diseases (n=39, 70.91%), and the major infectious pathogen was Mycoplasma (15, 38.46%). Conclusion Adenovirus HAI is prone to occur in infants under three years old, and can be secondary to infection caused by other respiratory pathogens such as Mycoplasma pneumoniae. Children with moderate to severe anemia, congenital heart disease, nervous system diseases and other underlying diseases are more likely to develop adenovirus HAI.