We have recently discovered the potential for in vivo naked plasmid DNA (pDNA) transfer into gastric serosal surface cells in mice. As pDNA are huge molecules, the mechanism of gene transfer without carriers and physical forces is of great biological interest. The endocytic route for naked pDNA transfer into gastric mesothelial cells was not clathrin- or caveolae-mediated endocytosis, but macropinocytosis. Naked pDNA transfer required both actin polymerization and myosin-based contraction. Upstream kinases of Rho family GTPases, Syk, Src family kinases and PI-3K were involved in naked pDNA transfer. Furthermore, the intracellular signaling pathway was not mediated via the Rho pathway, but by the Rac pathway. Downstream molecules of Rac, PAK and WAVE2 co-operated with naked pDNA transfer. Overall, it was demonstrated that the Rac signaling pathway regulated the macropinocytosis of naked pDNA. The information in this study would be helpful to clarify in vivo cell functions and to improve in vivo transfection efficiency.