Background <p>This study aimed to describe the epidemiological trends of <i>Mycoplasma pneumoniae</i> (MP) infection among children with acute respiratory tract infections (ARTIs) before, during and after the COVID-19 pandemic, and evaluating the impact of non-pharmaceutical interventions (NPIs) on the epidemiology of MP infection.</p> Methods <p>Children with ARTIs admitted to the Children’s Hospital of Soochow University (SCH) from January 2010 to December 2023 and underwent MP nucleic acid PCR assay were included. Clinical data on age, sex, onset time and detection result were collected and analyzed.</p> Results <p>All of the 122,984 inpatients were enrolled, in which 20.8% (25659/122984) of the children with MP tested positive, including 19.4% (14139/72662) for male and 22.9% (11520/50322) for female. It was a statistically significant difference between the two genders (<i>p</i> &lt; 0.05). In addition, the positive rate of MP was the highest in the age group &gt; 6 years old each year (<i>p</i> &lt; 0.05). During 14-year period, the detection rate of MP has experienced four peaks in 2012, 2013, 2019, and 2023. Before the NPIs the prevalence of MP showed seasonality, and the number and rate of MP positivity reached their peak in August. However, the rate of MP positivity remained at a low level during the NPIs. After the abolition of NPIs, the MP positivity rate obviously increased and remained at a high level.</p> Conclusions <p>The NPIs could reduce the spread of MP infection and change its epidemic season, but it has not changed the susceptible population of MP infection.</p>