Circular RNAs (circRNAs) regulate Jumonji domain-containing protein-3 (JMJD3) by sponging with microRNAs (miRNAs). This study aimed to investigate the role of icariin on specific circRNA/miRNA/JMJD3 axis in osteogenic differentiation of MC3T3-E1 cells. CircRNA sequencing was performed on the MC3T3-E1 cells induced by osteogenic differentiation medium for 1 d (negative control (NC) group) and 14 d (osteogenesis group). And mmu_circ_0000349 was verified using Sanger sequencing, ribonuclease R degradation, and actinomycin D assay. The function of mmu_circ_0000349 was validated by detecting the expressions of osteogenic differentiation markers, alkaline phosphatase (ALP), and runt-related transcription (RUNX2), via realtime quantitative PCR (qPCR) and Western blotting or ALP and alizarin red staining assay. Dual luciferase reporter gene assay confirmed the relationship between mmu_circ_0000349 and mmumiR-138-5p (or mmu-miR-138-5p and JMJD3). Meanwhile, the JMJD3 binding to mmu_circ_0000349 was screened using an RNA pull-down assay. qPCR and Western blotting confirmed the effect of icariin on the mmu_circ_0000349/mmu-miR-138-5p/JMJD3 axis and osteogenic differentiation. As MC3T3-E1 osteogenic differentiation progressed, the JMJD3 expression level increased. A total of 361 circRNAs exhibited differences between the NC and osteogenesis groups. After validation, mmu_circ_0000349 was further analyzed as it exhibited the largest expression. And mmu_circ_0000349 was identified as a stable circular structure. Overexpression of mmu_circ_0000349 increased the expression levels of ALP and RUNX2, enhanced ALP activity, and increased the number of mineralized nodules; contrarily, inhibition of mmu_circ_0000349 exerted opposite effects. The data also confirmed that mmu_circ_0000349 regulated JMJD3 by sponging with mmu-miR-138-5p. With the increase in icariin concentration and time for treatment, the expression levels of mmu_circ_0000349, JMJD3, ALP, and RUNX2 also increased, whereas that of mmu-miR-138-5p decreased. In conclusion, Icariin promoted osteogenic differentiation by regulating the mmu_circ_0000349/mmu-miR-138-5p/JMJD3 pathway. Therefore, this provides a theoretical basis for the treatment of diseases related to osteogenic differentiation.