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Objective To investigate the impact of transition from National Institute for Health and Care Excellence (NICE) guidelines to Kaiser Permanente Neonatal Sepsis Risk Calculator (KP-SRC) on early-onset sepsis (EOS) diagnosis and mortality.Study design Pre–post intervention analysis across 16 NHS hospital Trusts in England (2015–2023) 18 months before and after NICE to KP-SRC transition. Liveborn infants of any gestational age (N=222 122) were extracted from Hospital Episode Statistics and linked to national microbiological data and birth and death registrations.Main outcomes Cases were identified within first 3 days of life during birth admission or first 7 days if readmitted and defined microbiologically (positive microbiology from normally sterile site) or clinically (International Classification of Diseases, 10th Revision codes). Deaths occurring within the neonatal period attributable to EOS and proportion of EOS cases diagnosed during readmission were identified.Results There was no evidence of an increase in late diagnosis of EOS after KP-SRC implementation across hospitals combined, with the proportion of microbiologically confirmed cases diagnosed on readmission similar to pre-implementation (9.6% vs 10.8%; p=0.67). Variation between hospitals was observed, with non-significant increases in readmission diagnosis (vs diagnosis in birth episode) post implementation in 9 of 15. There was no difference in mortality for microbiologically confirmed cases or deaths based on clinical diagnosis though the study was not powered for these outcomes.Conclusion No overall increase in late diagnoses due to EOS was observed after switching to KP-SRC but variation between hospitals, including non-significant increases in readmission diagnoses in some, injects caution and underscores the importance of local monitoring post implementation.