Breastfeeding undoubtedly provides important benefits to the mother-infant dyad and should be encouraged. Mastitis, one of the common but major cause of premature weaning among lactating women, is an inflammation of connective tissue within the mammary gland. This study reports the influence of mastitis on human milk microbiota by utilizing 16 S rRNA gene sequencing approach. We sampled and sequenced microbiome from 50 human milk samples, including 16 subacute mastitis (SAM), 16 acute mastitis (AM) and 18 healthy-controls. Compared to controls, SAM and AM microbiota were quite distinct and drastically reduced. Genera including, Aeromonas, Staphylococcus, Ralstonia, Klebsiella, Serratia, Enterococcus and Pseudomonas were significantly enriched in SAM and AM samples, while Acinetobacter, Ruminococcus, Clostridium, Faecalibacterium and Eubacterium were consistently depleted. Further analysis of our samples revealed positive aerotolerant odds ratio, indicating dramatic depletion of obligate anaerobes and enrichment of aerotolerant bacteria during the course of mastitis. In addition, predicted functional metagenomics identified several gene pathways related to bacterial proliferation and colonization (e.g. two-component system, bacterial secretion system and motility proteins) in SAM and AM samples. In conclusion, our study confirmed previous hypothesis that mastitis women have lower microbial diversity, increased abundance of opportunistic pathogens and depletion of commensal obligate anaerobes.