Objective To investigate the protoplast preparation and transformation system of endophytic fungus Falciphora oryzae. Results F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9 M KCl solution and 10 mg mL(-1) glucanase at 30 degrees C with shaking at 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar medium containing 1 M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired green fluorescent protein-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots. Conclusions This study provided a protoplast transformation system of F. oryzae, creating opportunities for future genetic research in other endophytic fungi.