Abstract Background Wharton’s Jelly-derived mesenchymal stromal cells (WJ-MSCs) present several advantages over other sources of multipotent stem cells, not only because they are obtained from neonatal umbilical cord, which is considered a biological waste, but also display higher proliferation rate and low senescence at later passages compared to stromal cells obtained from other sources. In the field of tissue engineering, WJ-MSCs have a wide therapeutic potential, due to their multipotential capacity, which can be reinforced if cells are genetically modified to direct their differentiation towards a specific lineage; unfortunately, as primary cells, WJ-MSC are difficult to transfect. Therefore, the objective of the present work was to standardize a protocol for the transfection of WJ-MSCs using a cationic polymer. Such protocol is important for future developments that contemplate the genetic modification of WJ-MSCs for therapeutic purposes. Methods In this work, WJ-MSCs were genetically modified using polyethylenimine (PEI) and a lentiviral plasmid that encodes for green fluorescent protein (pGFP). To achieve WJ-MSCs transfection, complexes between PEI and pGFP, varying its composition (N/P ratio), were evaluated and characterized by size, zeta potential and cytotoxicity. At the N/P ratio condition where the highest transfection efficiencies were obtained, immunophenotype, immunomodulation properties and multipotential capacity of WJ-MSCs were evaluated. Results Here, we present the standardization of the transfection conditions of the WJ-MSCs in a monolayer culture with PEI. The concentrations of plasmid and PEI that have the best transfection efficiencies were established Conclusions Transfection with PEI doesn’t affect immunophenotype, immunomodulatory properties and differentiation capacity of WJ-MSCs.