Objective To understand the clinical characteristics, bacterial serotypes and antimicrobial resistance of invasive Streptococcus pneumoniae(S. pneumoniae) infection in hospitalized children in a hospital, guide clinical rational antimicrobial use and find effective methods for prevention and treatment of invasive S. pneumoniae infection. Methods Retrospective analysis was performed on clinical data, antimicrobial susceptibility resting results and serotype of S. pneumoniae as well as vaccine serotype coverage of children who were diagnosed with invasive S. pneumoniae infection in a hospital from January 2014 to December 2018. Results 74 children were with invasive S. pneumoniae infection, mainly children in early childhood (55.4%), the main infection site was blood (44.6%). Among 74 strains of S. pneumoniae, 22 were from cerebrospinal fluid, 13 serotypes were divided, the common serotypes were 19F, 19A, 14 and 23F. Isolation rates of penicillin-non-susceptible S. pneumoniae (PNSP) was 56.7% (42 strains), no strain was found to be resistant to linezolid, vancomycin, moxifloxacin and levofloxacin. Resistance rates of strains with different serotypes was significantly different, resistance rates of serotypes 19F and 19A to penicillin, cefotaxime, cefepime and meropenem were higher than other serotypes. The coverage of S. pneumoniae vaccine to S. pneumoniae was 63.5% in PCV7 and 97.3% in PCV13. Conclusion Invasive S. pneumoniae infection is more common in infants and young children in this hospital, serotypes of isolated S. pneumoniae are mainly 19F, 19A, 14 and 23A, PCV13 has a high coverage rate for these serotypes, which can reduce the occurrence of invasive S. pneumoniae infection through vaccination. Serotypes with high prevalence tend to show higher antimicrobial resistance, so antimicrobial agents should be selected rationally according to antimicrobial susceptibility testing results.