Irisin, a recently identified hormone secreted by skeletal muscle in response to exercise, exhibits anabolic effects on the skeleton primarily through the stimulation of bone formation. However, the mechanism underlying the irisin-stimulated anabolic response remains largely unknown. To uncover the underlying mechanism, we biosynthesized recombinant irisin (r-irisin) using an Escherichia coli expression system and used it to treat several osteoblast cell types. Our synthesized r-irisin could promote proliferation and differentiation of osteoblasts as evidenced by enhanced expression of osteoblast-specific transcriptional factors, including Runt-related transcription factor-2 (Runx2), Oster (Osx), as well as early osteoblastic differentiation markers such as alkaline phosphatase (Alp) and collagen type I alpha 1 (Col1a1). Furthermore, we showed that the promotion of r-irisin on the proliferation and differentiation of osteoblast lineage cells are preferentially through aerobic glycolysis, as indicated by the enhanced abundance of representative enzymes such as lactate dehydrogenase A (LDHA) and pyruvate dehydrogenase kinase 1 (PDK1), together with increased lactate levels. Suppression of r-irisin-mediated aerobic glycolysis with Dichloroacetate blunted its anabolic effects. The favorite of the aerobic glycolysis after r-irisin treatment was then confirmed in primary calvarial cells by metabolic analysis using gas chromatography-mass spectrometry. Thus, our results suggest that the anabolic actions of r-irisin on the regulation of osteoblast lineage cells are preferentially through aerobic glycolysis, which may help to develop new irisin-based bone anabolic agents.