The success of cancer immunotherapy through the adoptive transfer of cytotoxic T lymphocytes (CTLs) is highly dependent on the potency of the elicited anti-tumor responses generated by the transferred cells, which can be hindered by a variety of upregulated immunosuppressive pathways. Downregulation of these pathways in the T cells via RNA interference (RNAi) could significantly boost their capacity to infiltrate tumors, proliferate, persist, and eradicate tumor cells, thus leading to a durable anti-tumor response. Unfortunately, it is well known that primary T cells are hard-to-transfect and conventional non-viral transfection agents are generally ineffective. Viral transduction and electroporation are more efficient but their use is restricted by high cost, safety issues, and cytotoxicity. Photoporation has recently gained interest as a more gentle alternative physical approach to deliver membrane-impermeable macromolecules into cells. By attaching gold nanoparticles (AuNPs) to the cell surface followed by pulsed laser illumination, transient membrane pores can be generated that allow the diffusion of macromolecules directly into the cell cytosol. Here, we evaluated this technique for the non-toxic and effective delivery of small interfering RNA (siRNA) and subsequent silencing of target genes in activated CTLs. We compared photoporation with nucleofection, the current standard physical technique for T cell transfection, and demonstrated a significantly reduced cytotoxicity and higher average dose per cell for the photoporation technique.