: Recent evidence suggests that gut microbiome plays a role in the development of pancreatic ductal adenocarcinoma (PDAC) and influences treatment response. However, the association of tumor location and preoperative biliary stenting (PBS) with gut microbial composition and diversity remains poorly understood. : Preoperative stool specimens were prospectively collected from patients with PDAC undergoing surgery between March 2020 and July 2021 at the Department of Surgery, Heidelberg University Hospital, Germany. Whole-genome shotgun metagenomic sequencing was performed. Microbial diversity was assessed using the Shannon index and Bray-Curtis dissimilarity with principal coordinates analysis. : A total of 63 preoperative stool samples were analyzed from 40 patients with pancreatic head (63.5%) and 23 with body/tail tumors (36.5%). Baseline characteristics were comparable between groups. Microbial community composition differed significantly between tumor locations (Bray-Curtis, = 0.005), with enrichment of in body/tail tumors. Among patients with pancreatic head tumors, PBS was associated with reduced alpha diversity (Shannon index, = 0.04) and depletion of taxa including and , and members of the genera and . PBS was associated with a higher rate of major complications > 3a according to the Clavien-Dindo classification (28.6% vs. 3.8%; = 0.04). : PBS was associated with reduced microbial diversity and distinct taxonomic alterations of the gut microbiome. These findings suggest that biliary stenting is associated with microbiome alterations that may be relevant for perioperative risk stratification and warrant further investigation.