Objective Conduct in vitro absorption rate and zebrafish anti osteoporosis efficacy testing on YQJ calcium zinc vitamin D vitamin K oral solution (referred to as "YQJ") and its disassembled formula, and explore the potential mechanism based on transcriptome sequencing. Methods Using Caco-2 cells to simulate gastrointestinal absorption, compare the calcium ion absorption rates of YQJ and its disassembled formulas. Induction of osteoporosis model using dexamethasone, staining of bone density with Alizarin Red, and dual validation of drug efficacy using Tg (ola. sp7: nlsGFP) osteoblast fluorescence zebrafish. Set up YQJ 250 μg·mL-1, MC, and NC groups, each with three biological replicates, and perform transcriptome sequencing on zebrafish. Strictly screen the differentially expressed genes at the intersection of the three groups using | log2fold change | ≥ 2 and q-value ≤ 0.001, perform functional annotation and enrichment analysis, screen out the expression pathway of YQJ anti osteoporosis genes, and perform QPCR validation. Results The absolute absorption rate of YQJ is 42.6%, which is 133%~159% compared to the four different formulas, and 170% compared to single calcium citrate. The synergistic effect of YQJ on promoting calcium absorption is significant. YQJ can significantly restore zebrafish skull density, increase osteogenic fluorescence intensity, and at a concentration of 125 μg·mL-1, the anti osteoporosis effect of YQJ increased by 24% -25% compared to calcium citrate alone. The sequencing results showed 335 and 231 differentially expressed genes in MC/NC and YQJ/MC, with 58 overlapping genes at the intersection of the three groups. Functional enrichment of overlapping genes revealed that YQJ"s anti osteoporosis effect involves 4 pathways and 11 key genes. Conclusions The calcium absorption rate and anti osteoporosis effect of YQJ are superior to those of split formula and single calcium citrate; YQJ can increase skull density and osteoblast signaling intensity, mainly by regulating complement and coagulation cascades, ferroptosis, phagosome, response to oxidative stress signaling pathways, and other functional proteins, specifically involving the regulation of key genes c3a.1, c3a.2, c3a.6, c9, c5, cfb, coro1a, cp, mpx, cldni, and bmpr1aa, synergistically exerting anti osteoporosis effects.