The strategy of using conductive materials in regenerating bone defects is attractive, benefiting from the bioelectricity feature of natural bone tissues. Thereby, POP conductive fibers were fabricated by coating polypyrrole (PPY) onto electrospun poly(l-lactide) (PLLA) fibers, and their potentials in promoting osteogenic differentiation of bone mesenchymal stromal cells (BMSCs) were investigated. Different from the smooth-surfaced PLLA fibers, POP fibers were rough-surfaced and favorable for protein adsorption and mineralization nucleation. When electrical stimulation (ES) was applied, the surface charges on the conductive POP fibers further promoted the protein adsorption and the mineral deposition, while the non-conductive PLLA fibers displayed no such promotion. When BMSCs were cultured on these fibers, strong cell viability was detected, indicating their good biocompatibility and cell affinity. In osteogenic differentiation studies, BMSCs demonstrated the strongest ability in differentiating toward osteoblasts when they were cultured on the POP fibers under ES, followed by the case without ES. In comparison with the conductive POP fibers, the non-conductive PLLA fibers displayed significantly weaker ability in inducing the osteogenic differentiation of BMSCs with ES being applied or not. Alongside the differentiation, both the calcium deposition on BMSC/material complexes and the intracellular Ca concentration were identified the most abundant when BMSCs grew on the POP fibers under ES. These findings suggested that the surface charges of conductive fibers played roles in regulating protein adsorption, ion migration and nucleation, particularly under ES, which contributed much to the increased intracellular Ca ions, and thus accelerated the osteogenic differentiation of the seeded cells. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.