Irritable bowel syndrome (IBS) is a common disorder of gut-brain interaction with multifaceted pathophysiology. Prior studies have demonstrated higher rates of vitamin D deficiency in individuals with IBS compared to healthy controls (HC), as well as associations of vitamin D concentration with IBS symptoms. A systematic review of 10 mouse and 14 human studies reported a positive association between vitamin D (serum levels and supplementation) and beta diversity of gut microbiome in a variety of conditions. The present retrospective case-control study aimed to compare vitamin D (25(OH)D) plasma concentrations and gut microbiome composition in adult women with IBS (n=99) and HC (n=62). Plasma concentrations of 25(OH)D were assessed using the Endocrine Society Guidelines definition of vitamin D deficiency (25(OH)D <20 ng/ml) and insufficiency (25(OH)D >20-<30 ng/ml). 16S rRNA microbiome gene sequencing data was available for 39 HC and 62 participants with IBS. Genus-level Bifidobacterium and Lactobacillus and phylum-level Firmicutes and Bacteroidetes relative abundances were extracted from microbiome profiles. Results showed vitamin D deficiency in 40.3% (n=25) vs. 41.4% (n=41), and insufficiency 33.9% (n=21) vs. 34.3% (n=34) in the HCs vs. IBS groups, respectively. The odds of IBS did not differ depending on 25(OH)D status (p=0.75 for deficient, p=0.78 for insufficient), and the average plasma vitamin D concentration did not differ between IBS (mean 24.8 ng/ml) and HCs (mean 25.1 ng/ml; p=0.57). We did not find evidence of an association between plasma 25(OH)D concentration and richness, Shannon index, Simpson index or specific bacterial abundances in either HCs or the IBS group.