Background <p>Nonviral gene delivery using DNA vectors is widely used in cell engineering, vaccination, and gene therapy, but delivery efficiency remains lower than those of viral vectors and mRNA-based approaches, partly due to inefficient nuclear entry, as transfected DNA must enter the nucleus for transcription. Therefore, a mechanistic understanding of nuclear entry pathways is essential for developing strategies to improve the efficiency.</p> Methods <p>This review evaluated mechanistic studies of DNA nuclear entry in mammalian cells, mathematical models of intracellular DNA trafficking, quantitative analyses of DNA nuclear accumulation and transgene expression, and strategies to enhance nuclear delivery of DNA.</p> Results <p>Two mechanistically distinct pathways for DNA nuclear entry have been reported: enclosure upon nuclear envelope reformation in divided cells, and active transport through nuclear pore complexes (NPCs). Various strategies have been developed to enhance nuclear import through these pathways; however, their effectiveness depends on multiple factors, including cell type, delivery methods, and cell cycle status. Although DNA vectors are significantly larger than the nominal inner diameter of NPCs, they may traverse NPCs through deformation and interactions with nuclear transport proteins. Quantitative studies show that DNA nuclear accumulation is time dependent and heterogeneous within the same population.</p> Conclusions <p>Nuclear entry plays a key role in determining efficiency of nonviral gene delivery. Advances in mechanistic studies, quantitative modeling, and imaging-based analyses have improved our understanding of intracellular DNA trafficking and nuclear accumulation. Integrating these insights with delivery strategies that enhance nuclear access while preserving the cellular machinery required for transgene expression will be critical for developing more efficient and reliable nonviral DNA delivery systems for therapeutic and biotechnological applications.</p>