Purpose Our study was designed to examine the possible relationship between gut microbiota, sleep disturbances, and acute postoperative pain. Methods Using 16S rRNA sequencing, we analyzed preoperative fecal samples from women undergoing breast cancer surgery. Preoperative sleep disturbance was evaluated with the Pittsburgh Sleep Quality Index (PSQI) questionnaire. Peak and average pain at rest and movement were evaluated 24 h after surgery, using a numerical rating scale (NRS). Preoperative symptoms of depression and anxiety were assessed with the Patient Health Questionnaire-9 (PHQ-9) and Generalized Anxiety Disorder-7 (GAD-7), respectively. Inflammation was measured using white blood cell and neutrophil counts, together with platelet-lymphocyte ratio, and neutrophil-lymphocyte ratio. Results Preoperative sleep disturbance was associated with more severe acute postoperative pain. At the phylum level, women with poor sleep quality had higher relative abundance of Firmicutes ( p = 0.021) and lower relative abundance of Bacteroidetes ( p = 0.013). At the genus level, women with poor sleep quality harbored higher relative abundance of Acidaminococcus and lower relative abundance of several genera. The genus Alloprevotella was negatively associated with peak pain at movement during the first 24 h ( r = − 0.592, p < 0.001). The genus Desulfovibrio was negatively associated with symptoms of anxiety ( r = − 0.448, p = 0.006). However, partial correlations suggested that the relationship between Alloprevotella and peak pain at movement during the first 24 h was not statistically significant after controlling for sleep ( r = − 0.134, p = 0.443). Conclusion These findings suggest that the changed gut microbiota may be involved in sleep-pain interaction and could be applied as a potential preventive method for postoperative pain. Trial registration The present clinical study has been registered on Chinese Clinical Trial Registry ( www.chictr.org.cn ); the clinical trial registration number is ChiCTR1900021730; the date of registration is March 7, 2019.