Background Host immune responses during late-onset sepsis (LOS) in very preterm infants are poorly characterised due to a complex and dynamic pathophysiology and challenges in working with small available blood volumes. We present here an unbiased transcriptomic analysis of whole peripheral blood from very preterm infants at the time of LOS. Methods RNA-Seq was performed on peripheral blood samples (6–29 days postnatal age) taken at the time of suspected LOS from very preterm infants <30 weeks gestational age. Infants were classified based on blood culture positivity and elevated C-reactive protein concentrations as having confirmed LOS (n = 5), possible LOS (n = 4) or no LOS (n = 9). Bioinformatics and statistical analyses performed included pathway over-representation and protein-protein interaction network analyses. Plasma cytokine immunoassays were performed to validate differentially expressed cytokine pathways. Results The blood leukocyte transcriptional responses of infants with confirmed LOS differed significantly from infants without LOS (1,317 differentially expressed genes). However, infants with possible LOS could not be distinguished from infants with no LOS or confirmed LOS. Transcriptional alterations associated with LOS included genes involved in pathogen recognition (mainly TLR pathways), cytokine signalling (both pro-inflammatory and inhibitory responses), immune and haematological regulation (including cell death pathways), and metabolism (altered cholesterol biosynthesis). At the transcriptional-level cytokine responses during LOS were characterised by over-representation of IFN-α/β, IFN-γ, IL-1 and IL-6 signalling pathways and up-regulation of genes for inflammatory responses. Infants with confirmed LOS had significantly higher levels of IL-1α and IL-6 in their plasma. Conclusions Blood responses in very preterm infants with LOS are characterised by altered host immune responses that appear to reflect unbalanced immuno-metabolic homeostasis.