Probiotics display estrogen-upregulating properties, yet the underlying mechanisms remain unclear. Herein, we investigated the therapeutic effects of five Bifidobacterium adolescentis strains on ovariectomized (OVX) rats. Among them, strains FHNFQ27M3 (CCFM1506) and FJSWXJNDX6M19 (CCFM1507) administration in OVX rats exhibited significant protective effects through increasing estrogen level and ameliorating pathological parameters, respectively. While FHNFQ27M3 significantly reduced appetite and alleviated abdominal obesity. Both effective strains (FHNFQ27M3 and FJSWXJNDX6M19) significantly altered the gut microbial composition by modifying the Firmicutes/Bacteroidetes ratio and increasing the abundances of Streptococcus, which was associated with elevated production of the short-chain fatty acid. Furthermore, serum metabolomic profiling revealed enhanced amino acid biosynthesis, with tryptophan concentration showed a significant negative correlation with estrogen level. Mechanistically, both strains promoted estrogen biosynthesis through upregulating CYP19A1 activity in adrenal glands. This study provided new insights into the gut microbiota-estrogen axis and highlight the potential of probiotic-mediated metabolic modulation as a viable therapeutic strategy for estrogen deficiency-related disorders.