This study investigated the intervention ability and potential mechanism of Lactobacillus helveticus-derived whey on ovariectomy (OVX)-induced osteoporosis. The whey from two L. helveticus strains, ATCC15009, and CCFM1096, were orally gavaged to OVX rats at a dose of 500 mg/kg for 12 weeks. The effect of L. helveticus-derived whey on osteoporosis varied, whereas the raw milk-derived whey showed no significant effect. CCFM1096 whey significantly enhanced bone biomechanics, improved bone microstructure, upregulated bone formation markers (bone alkaline phosphatase (BALP), bone gamma-carboxyglutamic acid-containing protein (BPG)), and downregulated bone resorption markers (tartrate-resistant acid phosphatase (TRAP), C-terminal telopeptide of type Ⅰ collagen (CTX-1), procollagen type Ⅰ N-terminal propeptide (PINP)), leading to promotion of osteoblast formation and inhibition of osteoclast generation. CCFM1096 whey affected bile acid metabolism, restored intestinal homeostasis, and prevented osteoporosis in OVX rats, with higher concentrations of unsaturated fatty acids, orotic acid, and uridine than that of ATCC15009. These findings provide important insights for the development of functional dairy products targeting osteoporotic populations and further demonstrate the importance of starter cultures in fermented foods.