INTRODUCTION: Previous research suggests that clodronate, a vesicular nucleotide transporter inhibitor, may suppress intestinal failure-associated liver disease (IFALD) in short bowel syndrome (SBS). The present study investigated the effects of clodronate on the gut microbiota and colonic barrier in SBS rat model. PURPOSE: Sprague-Dawley rats underwent 90% small bowel resection and then were randomized into three groups (n = 8 each): SBS/TPN (Control), SBS/TPN with low-dose clodronate (Low, 20 mg/kg/day), and SBS/TPN with high-dose clodronate (High, 60 mg/kg/day). After 7 days, fecal and colon samples were collected to analyze the gut microbiota, histological findings, gene expression of tight junction, and inflammatory markers. RESULTS: While alpha diversity showed no significant differences between the groups, high-dose clodronate-induced heterogeneity in the bacterial community structure. At the phylum level, the Firmicutes/Bacteroidota ratio significantly decreased in the High group. The crypt depth at distal colon was significantly deepest in the High group (p = 0.01). The Claudin-1 expression decreased significantly in the Low group in proximal colon (p = 0.019). The NLRP3 and IL-6 expressions in proximal colon showed significant differences among the groups (NLRP3: p < 0.001, IL-6: p = 0.021). CONCLUSION: Clodronate-induced alterations in the gut microbiota may contribute to IFALD suppression, albeit with potentially adverse effects on the colonic barrier.