OBJECTIVE Neonatal meningitis (NM) caused by Escherichia coli remains a major health problem in industrialized countries. Currently, information on the epidemiology and antimicrobial susceptibility patterns of NM in developing countries such as China is relatively scarce. Therefore, the present study investigated changes in the antimicrobial susceptibility of E. coli causing NM in a perinatal center in eastern China over the past 20 years. METHODS This survey was conducted during three periods: 2001-2006, 2007-2012, and 2013-2020. NM was diagnosed according to the number of white blood cells in the cerebrospinal fluid (CSF) and the presence of a single potential pathogenic bacterium in the culture prepared from the blood or CSF of a newborn baby. Changes in the antimicrobial susceptibility of E. coli were analyzed. RESULTS In total, 182 NM cases were identified. E. coli was identified in 69 of these cases, and in 21 of these cases, extended-spectrum beta-lactamase (ESBL) production was detected. E. coli was the main cause of NM identified in this study. The overall susceptibility of E. coli to third-generation cephalosporins such as cefotaxime decreased from 100% during 2001-2006 to 50% during 2007-2012 and, subsequently, increased to 71.0% during 2013-2020. This pattern of change is correlated with bacterial ESBL production. Only 8.3% of E. coli found in samples collected from infants with early onset meningitis (EOM) produced ESBL, while 37.3% of E. coli isolated from children with late-onset meningitis (LOM) produced ESBL. CONCLUSIONS E. coli remains the primary pathogen of NM. Compared with that isolated from infants with LOM, the percentage of ESBL-producing multidrug-resistant E. coli isolated from infants with EOM is significantly lower. Clinicians should consider this trend when determining appropriate and effective antibiotics as empirical treatment for NM.