Direct current (DC) stimulation has been used to promote bone repair and osteogenesis, but problems associated with the implanted metal electrodes may limit its application and compromise the therapeutic results. The replacement of the metal electrodes with a biodegradable conductive polymer film can potentially overcome these problems. In our work, polypyrrole/chitosan films comprising polypyrrole nanoparticles dispersed in a chitosan matrix were prepared. The polypyrrole/chitosan film meets the requirements for DC delivery, as indicated by its electrical conductivity, biodegradability, and mechanical properties. The film supports osteoblast growth to the same degree as dentine discs (a bone-like mineralized substrate), confirming that it is non-cytotoxic. Our results showed that optimal DC stimulation was achieved with 200 mu A for 4h per day, and under this condition, osteoblast metabolic activity on Day 7 increased by 1.8-fold over that without DC stimulation. To further improve osteogenesis on the polypyrrole/chitosan film, bone morphogenetic protein-2 (BMP-2) was covalently immobilized on the film surface. Osteoblasts cultured on the BMP-2-functionalized polypyrrole/chitosan film and subjected to the optimal DC stimulation exhibited a significant increase in cellular metabolic activity (2.3-fold on Day 7), ALP activity (1.7-fold on Day 21) and mineralization (twofold on Day 21) over those cultured on polypyrrole/chitosan film without DC stimulation. Osteogenic gene expression results showed that BMP-2 and DC stimulation by itself enhanced osteoblast differentiation, and a combination of these two factors resulted in synergistic effects on osteoblast differentiation and maturation. Biotechnol. Bioeng. 2013; 110: 14661475. (c) 2012 Wiley Periodicals, Inc.