BACKGROUND: Whether pathophysiological factors differ between males and females with irritable bowel syndrome-diarrhea (IBS-D) remains to be tested. To better understand potential sex differences, males with IBS-D were compared to naturally cycling females and to females with IBS-D taking hormonal contraception on plasma levels of cytokines and gut microbiome characteristics. METHODS: Males and females with Rome III IBS-D completed questionnaires and kept a daily symptom diary for 28 days. Blood and stool samples were collected between days 3 and 8 of the daily diary (estrogen-dominant days in naturally cycling females). Blood samples were analyzed for lipopolysaccharide (LPS)-stimulated and unstimulated cytokine levels. Stool samples were analyzed for microbiota signatures using 16S rRNA sequencing. RESULTS: Forty-seven participants with IBS-D (13 males, 22 naturally cycling females, 12 females with hormonal contraception use) ages 18 to 50 years were studied. Males had similar unstimulated IL10, IL12P40, IL12P70, IL1β, IL8, and TNFα plasma cytokine levels compared to naturally cycling females, but higher levels compared with females using hormonal contraception. LPS-stimulated IL12P70 levels were lower in both groups of females vs. males. Alpha- and beta-diversity did not differ although differences in genus-level bacteria were found. CONCLUSION: Cytokine levels differed between males and females using hormonal contraceptives but not between males and normally cycling females. It is important to consider that naturally cycling females may have a different cytokine and microbiome profile than females using hormonal contraceptives. Whether this portends a sex difference in potential etiologic factors remains to be determined.