Background:Due to the high prevalence and complex etiology, bovine mastitis (BM) is one of the most important diseases to compromise dairy cow health and milk quality. The shift in milk compositions has been widely investigated during mastitis, but recent studies suggested that gastrointestinal microorganism also has a crucial effect on the inflammation of other peripheral tissues and organs, including the mammary gland. However, research focused on the variation of rumen inner-environment during mastitis is still limited. Therefore, the ruminal microbial profiles, metabolites, and milk compositions in cows with different udder health conditions were compared in the present study. Furthermore, the correlations between udder health status and ruminal conditions were investigated. Based on the somatic cell counts (SCC), California mastitis test (CMT) parameters and clinical symptoms of mastitis, 60 lactating Holstein dairy cows with similar body conditions (excepted for the udder health condition) were randomly divided into 3 groups (n=20 per group) including the healthy (H) group, the subclinical mastitis (SM) group and the clinical mastitis (CM) group. Lactation performance and rumen fermentation parameters were recorded. And rumen microbiota and metabolites were also analyzed via 16S rRNA amplicon sequencing and untargeted metabolomics, respectively. Results:As the degree of mastitis increased, rumen lactic acid (LA) (P<0.01), acetate, propionate, butyrate, valerate (P<0.001), and total volatile fatty acids (TVFAs) (P<0.01) concentrations were significantly decreased. In the rumen of CM cows, the significantly increased bacteria related to intestinal and oral inflammation, such as Lachnospiraceae (FDR-adjusted P=0.039), Moraxella (FDR-adjusted P=0.011) and Neisseriaceae (FDR-adjusted P=0.036), etc., were accompanied by a significant increase in 12-oxo-20-dihydroxy-leukotriene B4 (FDR-adjusted P=5.97 × 10?9) and 10beta-hydroxy-6beta-isobutyrylfuranoeremophilane (FDR-adjusted P=3.88 × 10?10). Meanwhile, in the rumen of SM cows, the Ruminiclostridium_9 (FDR-adjusted P=0.042) and Enterorhabdus (FDR-adjusted P=0.043) were increased along with increasing methenamine (FDR-adjusted P=6.95 × 10?6), 5-hydroxymethyl-2 furancarboxaldehyde (5-HMF) (FDR-adjusted P=2.02 × 10?6) and 6-methoxymellein (FDR-adjusted P=2.57 × 10?5). The short-chain fatty acids (SCFAs)-producing bacteria and probiotics in rumen, including Prevoterotoella_1 (FDRadjusted P=0.045) and Bifidobacterium (FDR-adjusted P=0.035), etc., were significantly reduced, with decreasing 2phenylbutyric acid (2-PBA) (FDR-adjusted P=4.37 × 10?6). Conclusion:The results indicated that there was a significant shift in the ruminal microflora and metabolites associated with inflammation and immune responses during CM. Moreover, in the rumen of cows affected by SM, the relative abundance of several opportunistic pathogens and the level of metabolites which could produce antibacterial compounds or had a competitive inhibitory effect were all increased.