Substantial evidence indicates that pulsed electromagnetic fields (PEMF) could accelerate fracture healing and enhance bone mass, whereas the unclear mechanism by which PEMF stimulation promotes osteogenesis limits its extensive clinical application. In the present study, effects and potential molecular signaling mechanisms of PEMF on in vitro osteoblasts were systematically investigated. Osteoblast-like MC3T3-E1 cells were exposed to PEMF burst (0.5, 1, 2, or 6 h/day) with 15.38 Hz at various intensities (5 Gs (0.5 mT), 10 Gs (1 mT), or 20 Gs (2 mT)) for 3 consecutive days. PEMF stimulation at 20 Gs (2 mT) for 2 h/day exhibited most prominent promotive effects on osteoblastic proliferation via Cell Counting kit-8 analyses. PEMF exposure induced well-organized cytoskeleton, and promoted formation of extracellular matrix mineralization nodules. Significantly increased proliferation-related gene expressions at the proliferation phase were observed after PEMF stimulation, including Ccnd 1 and Ccne 1. PEMF resulted in significantly increased gene and protein expressions of alkaline phosphatase and osteocalcin at the differentiation phase of osteoblasts rather than the proliferation phase via quantitative reverse transcription polymerase chain reaction and Western blotting analyses. Moreover, PEMF upregulated gene and protein expressions of collagen type 1, Runt-related transcription factor 2 and Wnt/β-catenin signaling (Wnt1, Lrp6, and β-catenin) at proliferation and differentiation phases. Together, our present findings highlight that PEMF stimulated osteoblastic functions through a Wnt/β-catenin signaling-associated mechanism and, hence, regulates downstream osteogenesis-associated gene/protein expressions. Bioelectromagnetics. 37:152-162, 2016. © 2016 Wiley Periodicals, Inc.