Objective - Probiotics are live microbial supplements that colonize the gut and potentially exert health benefit to the host. We aimed to determine the impact of probiotic (Infloran®: Lactobacillus acidophilus-NCIMB701748 and Bifidobacterium bifidum-ATCC15696) on the bacterial and metabolic function of the preterm gut while on the neonatal intensive care unit (NICU) and following discharge. Patients - Stool samples (n = 88) were collected before, during, and after probiotic intake from 7 patients, along with time-matched controls from 3 patients. Samples were also collected following discharge home from the NICU. Methods - Samples underwent bacterial profiling analysis by 16S rRNA gene sequencing and quantitative PCR (qPCR), as well as metabolomics profiling using liquid chromatography mass spectrometry (LCMS). Results - Bacterial profiling showed greater Bifidobacterium (15.1%) and Lactobacillus (4.2%) during supplementation compared to the control group (4.0% and 0%, respectively). While Lactobacillus reduced after probiotic was stopped, Bifidobacterium remained high following discharge, suggestive of successful colonisation. qPCR analysis showed a significant increase (P = <0.01) of B. bifidum in infants who received probiotic treatment compared to controls, but no significant increase was observed for L. acidophilus (P = 0.153). Metabolite profiling showed clustering based on receiving probiotic or matched controls, with distinct metabolites associated with probiotic administration. Conclusions - Probiotic species successfully colonise the preterm gut, reducing the relative abundance of potentially pathogenic bacteria, and affecting gut functioning. Bifidobacterium (but not Lactobacillus) colonized the gut long-term; suggesting the possibility that therapeutically administered probiotics may continue to exert important functional effects on gut microbial communities in early infancy.