Transient gene expression in plant protoplasts is a powerful tool for analyzing gene function and for performing biotechnical manipulations. Here we-report the isolation of viable protoplastsfrom the fruit flesh of sweet cherry (Prunus avium L.) cv. Hong Deng and their polyethylene glycol (PEG)-mediated transient transfection using green fluorescent protein (GFP) as a marker gene. We investigated the main factors affecting the efficacy of protoplast isolation and transfection, including the composition of the enzymolysis solution, enzymolysis time, pH of the enzymolysis solution, PEG concentration, and transfection time. Protoplast isolation was optimal when the tissue was incubated in enzymolysis solution composed of 1.0% Cellulase R-10, 0.5% Pectolase Y-23, and 0.6 M mannitol (pH 5.8) for 18 h, resulting in a protoplast yield of 4.3 x 10(6) protoplasts/g fresh weight [FW] and viability of 84.1%. Protoplast transformation efficiency was measured by transient expression of the GFP reporter gene, and transformation efficiency was highest when protoplasts were incubated in transfection medium containing 40% PEG for 15 min. Collectively, this work describes an efficient protoplast isolation and protoplast transient expression system that can be used to facilitate molecular biology research in sweet cherry. (C) 2016 Elsevier B.V. All rights reserved.