The use of cell-penetrating peptides as transduction vectors is a promising approach to deliver peptides and proteins into cells. However, the uptake and bioavailability of trans-activating transcriptor (TAT)-conjugated molecules vary depending on the cell type and the cargo. This study aimed to determine whether a low-voltage electrical pulse can enhance the TAT-mediated delivery of peptide cargoes in different cell types. In TF-1 and mouse embryonic stem cells, the uptake of a novel detachable TAT-conjugated glycogen synthase kinase-3 (GSK-3) peptide inhibitor was enhanced by an order of magnitude without affecting the cell viability. A similar increase in uptake was achieved in primary mouse bone marrow cells while maintaining >80% of their viability. Interestingly, under these low-voltage conditions, the uptake of a control peptide not conjugated to TAT was not significantly increased. A T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase reporter assay was also used to assess the bioactivity of the TAT construct. The results indicated that cells loaded with a low-voltage electrical pulse had a twofold increase in TCF/LEF activity, which was equivalent to a level of GSK-3 inhibition similar to that of cells treated with 20 mmol/l lithium or 500 nmol/l (2'Z,3'E)-6-bromoindirubin-3'-oxime. These results demonstrate the usefulness of low-voltage electrical pulses to enhance the uptake and bioactivity of TAT-conjugated molecules in different cell types.