The ability to modulate deregulated genes by RNAi provides treatment perspectives in certain diseases including cancers. Electrotransfer of oligonucleotides was studied in vitro, showing a direct transfer of negatively charged siRNA across the plasma membrane into the cytoplasm. In vivo, the feasibility of siRNA electrotransfer was demonstrated in different studies and tissues. While effective, electrotransfer of siRNA into 3D tissues still needs to be understood. Here, we evaluated the efficiency of siRNA electrotransfer and assessed its effect in 3D spheroids made of HCT116-GFP cells by confocal fluorescence microscopy and flow cytometry. Our results indicate that siRNA uptake was not uniform across 3D multicellular spheroids. The electrophoretic migration of nucleic acids upon delivery of unipolar electric field pulses could explain the asymmetry of siRNA uptake. Moreover, a gradient was observed from external layers toward the center, leading to siRNA silencing of GFP positive cells located in the outer rim. While siRNA delivery experiments on spheroids may differ from intratumoral injections, the levels of transfection in spheroids are comparable to levels observed in published studies in vivo. Taken together, our results provide fundamental information about siRNA 3D distribution during electrotransfer, indicating that multicellular spheroids remain a relevant alternative to animal experimentation.