Abstract: Objective: To explore the effect of the combined application of lycopene, lutein and phytosterols on gut microbiota in postmenopausal osteoporosis mice. Methods: A postmenopausal osteoporosis model was established by bilateral ovariectomy (OVX) method. Sixty 12-week-old female C57BL/6J mice were randomly divided into sham operation group (sham group), OVX group, lycopene, lutein and phytosterols combined intervention with low (L), medium (M) and high (H) dose groups and estradiol valerate group (E2 group). They were administered by gavage 1 time per day for 12 weeks. MicroCT scans were used to analyze the changes of femoral bone mineral density (BMD) and bone microstructure in mice. Hematoxylin-eosin (HE) staining was used to observe the histopathological changes of femur in each group. Enzyme-linked immunoassay (ELISA) was used to detect the expression of osteocalcin (BGP) and tartrate-resistant phosphatase 5b (TRAP5b) in the femur. 16S rDNA amplicon sequencing was used to detect and analyze the microbiota genes in V3-V4 region in feces. Results: The HE results showed that the cancellous bone structure of the mice in the OVX group was changed, with sparse and loose trabecular bone, uneven thickness, a large number of fractures, and widened bone marrow cavity. The bone structure of the M and H groups was improved, and the H group was close to the sham group. Micro-CT results showed that compared with the sham group, the BMD, trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), connectivity density (Conn.Dn) in the OVX group were significantly decreased, and bone surface density (BS/TV), trabecular separation (Tb.Sp), structural model index (SMI) were significantly increased. Compared with the OVX group, BMD, BV/TV, BS/TV, Tb.N, and Conn.Dn in the M and H groups were significantly increased, while Tb.Sp and SMI in the H group were significantly decreased. The results of ELISA showed that compared with the OVX group, the BGP in the M and H groups was significantly increased, and TRAP5b was significantly decreased. The results of 16S rDNA amplicon sequencing showed that at the phylum level, the relative abundance of Verrucomicrobia in the H group was significantly higher than that in the OVX group, and the relative abundance of Bacteroidetes was lower than that in the OVX group. At the genus level, the relative abundances of Akkermansia, Lachnospiraceae_NK4A136, Bifidobacterium, Ileibacterium, and Parasutterella in the H group were higher than those in the OVX group, while those of Duneiella, Lactobacillus, Helicobacter and Alistipes were lower than those in the OVX group. The results of linear discriminant analysis effect (LEfSe) analysis showed that the Actinobacteria, unidentified Actinomycetes, Bifidobacteriales, Bifidobacterium, Bifidobacteriaceae, Ileibacterium valens and Ileibacterium were enriched in the H group. Conclusion: The combined application of lycopene, lutein and phytosterols can significantly improve the bone microstructure and alleviate bone loss in OVX mice, and the mechanism may be related to the regulation of gut microbiota structure and abundance.