Irritable bowel syndrome (IBS) is a heterogeneous condition with multifactorial pathogenesis. We studied deeply phenotyped individuals with microbiota sequencing enrolled in the American Gut Project. The IBS subjects were matched by age, gender, body mass index, geography, and dietary patterns with non-IBS controls. A total of 942 subjects with IBS-Diarrhea (IBS-D), IBS-Constipation (IBS-C), unclassified IBS (IBS-U), and 942 non-IBS controls were included. We compared taxonomic and functional composition of gut microbiota based on 16S sequencing data and linked them with clinical characteristics and dietary factors. Subjects with IBS-D or IBS-U but not IBS-C showed significantly reduced bacterial diversity (Shannon; p < .01). Distinct bacterial signatures were associated with different IBS subtypes, and the related functional changes were related to IBS pathogenesis, such as the increased hydrogen sulfide production pathway in IBS-D and the increased palmitoleate biosynthesis pathway in IBS-C. IBS subjects with depression showed lower abundance of and higher abundance of than those without depression. The relative abundance of microbial short-chain fatty acid production pathways was significantly lower in IBS patients with depression than those without depression in all three subtypes. Female, younger age in IBS-D, and older age in IBS-C were associated with more severe microbiota dysbiosis, and distinct dietary factors had significant effects on the gut microbiota in different IBS subtypes. Our analysis identified the compositional uniqueness of gut microbiota in different IBS subtypes. Distinct associations of the gut microbiota with depression in IBS provide insights into shared pathways in disease pathogenesis. These findings highlight the importance of personalized gut microbiome modulation approaches in different subtypes for optimal therapeutic effects.