Persistent post-surgical pain (PPSP) is defined as pain which continues after a surgical operation in a significant form for at least three months (and is not related to pre-existing painful conditions). PPSP is a common, under-recognised, and important clinical problem which affects millions of patients worldwide. Preventative measures which are currently available include the selection of a minimally invasive surgical technique and an aggressive multimodal perioperative analgesic regimen. More recently, a role for the gut microbiota in pain modulation has become increasingly apparent. This study aims to investigate any relationship between the gut microbiota and PPSP. A prospective observational study of 68 female adult patients undergoing surgery for management of breast cancer was carried out. Stool samples from 45 of these patients were obtained to analyse the composition of the gut microbiota. Measures of pain and state-trait anxiety were also taken to investigate further dimensions in any relationship between the gut microbiota and PPSP. At 12 weeks postoperatively, 21 patients (51.2%) did not have any pain and 20 patients (48.8%) reported feeling pain that persisted at that time. Analysis of the gut microbiota revealed significantly lower alpha diversity (using three measures) in those patients reporting severe pain at the 60 min post-operative and the 12 weeks post-operative timepoints. A cluster of taxa represented by Bifidobacterium longum, and Faecalibacterium prausnitzii was closely associated with those individuals reporting no pain at 12 weeks postoperatively, while Megamonas hypermegale, Bacteroides pectinophilus, Ruminococcus bromii, and Roseburia hominis clustered relatively closely in the group of patients fulfilling the criteria for persistent post-operative pain. We report for the first time specific associations between the gut microbiota composition and the presence or absence of PPSP. This may provide further insights into mechanisms behind the role of the gut microbiota in the development of PPSP and could inform future treatment strategies.