ObjectiveThe purpose of this study was to develop a method for the isolation, culture, and PEG-mediated protoplast transfection from leaves of in vitro-grown plants ofRicinus communis.ResultsFactors such as the enzymatic composition and the incubation time were evaluated. The enzymatic solution, containing 1.6% Cellulase-R10 and 0.8% Macerozyme-R10, with 16 h of incubation, was the best condition to achieve a high protoplast yield (481.16 × 104protoplasts/g FW) with a high percentage of viability (95%). The combination and concentration of enzymes have been shown to affect the protoplast isolation efficiency significantly. Furthermore, we found that a higher number of protoplasts (8.5 × 105protoplast/g FW) was obtained at a longer incubation time, but their viability decreased. We obtained a simple and efficient protocol to isolate protoplast fromRicinus communisleaves and culture. A PEG-mediated protoplast transfection protocol was also established to introduce plasmid DNA intoRicinus communisgenotypes cultivated in Colombia. Thus, strengthening advances in the genetic improvement processes for this crop are presented.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13104-023-06414-y.