Vehicle solutions used for intraperitoneal injection are generally regarded as inert, yet their effects on the gut microbiome have not been systematically evaluated. We examined the impact of four commonly used vehicle solutions—PEG, Ethanol, Tween, and DMSO—on the murine gut microbiome using whole-genome shotgun sequencing of fecal samples. Vehicle-treated mice showed significant alterations in microbial diversity, taxonomic composition, and functional pathway profiles compared with controls. Functional analyses revealed treatment-specific metabolic shifts, with the greatest number of altered pathways detected in the Tween group. Affected pathways included carbohydrate, amino acid, nucleotide, lipid, and fermentation-related metabolism. These findings indicate that commonly used intraperitoneal vehicle solutions can significantly influence gut microbial ecology and function. Vehicle choice should therefore be recognized as a critical experimental factor in microbiome-sensitive animal studies.