In order to clarify the mechanism of action of licorice flavonoids in alleviating bone loss caused by osteoporosis, this study compared the effects of four glycyrrhiza flavonoids, naringenin, liquiritigenin, isoliquiritigenin, and licochalcone A, on osteogenic differentiation and mineralization by molecular docking simulation, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content assays, and Runt-related transcription factor 2 (Runx2) expression, and explored their potential molecular mechanisms. The results of molecular docking showed that the docking score of liquiritigenin with the estrogen receptor (ER) was the highest. All four flavonoids up-regulated ALP activity and OCN concentration in MC3T3-E1 cells, thereby elevating the mineralization level, among which liquiritigenin was the most effective. Moreover, treatment with a phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002) inhibited liquiritigenin from inducing increased phosphorylation levels in the PI3K/protein kinase B (AKT) signaling pathway and up-regulation of Runx2 expression, suggesting that PI3K and AKT were involved in osteogenic action. Liquiritigenin reversed bone mineral density loss in a zebrafish osteoporosis model. These findings suggest that liquiritigenin has the most significant osteogenic effect among the four estrogen-like flavonoids, stimulating osteoblast differentiation and bone mineralization through the activation of Runx2 via the PI3K/AKT signaling pathways. In conclusion, this study highlights the great potential of liquiritigenin for preventing and treating osteoporosis.