In Western countries, breast cancer tends to occur in older postmenopausal women. However, in Asian countries, the proportion of younger premenopausal breast cancer patients is increasing. Increasing evidence suggests that the gut microbiota plays a critical role in breast cancer. However, studies on the gut microbiota in the context of breast cancer have mainly focused on postmenopausal breast cancer. Little is known about the gut microbiota in the context of premenopausal breast cancer. This study aimed to comprehensively explore the gut microbial profiles, diagnostic value, and functional pathways in premenopausal breast cancer patients. Here, we analyzed 267 breast cancer patients with different menopausal statuses and age-matched female controls. The α-diversity was significantly reduced in premenopausal breast cancer patients, and the β-diversity differed significantly between breast cancer patients and controls. By performing multiple analyses and classification, 14 microbial markers were identified in the different menopausal statuses of breast cancer. Bacteroides fragilis was specifically found in young women of premenopausal statuses and Klebsiella pneumoniae in older women of postmenopausal statuses. In addition, menopausal-specific microbial markers could exhibit excellent discriminatory ability in distinguishing breast cancer patients from controls. Finally, the functional pathways differed between breast cancer patients and controls. Our findings provide the first evidence that the gut microbiota in premenopausal breast cancer patients differs from that in postmenopausal breast cancer patients and shed light on menopausal-specific microbial markers for diagnosis and investigation, ultimately providing a noninvasive approach for breast cancer detection and a novel strategy for preventing premenopausal breast cancer.