Rath peptide has been introduced as a delivery vector that transports various membrane-impermeable cargoes in a non-covalent fashion. In this paper, we present a study on Rath peptide conjugated with fluorescein-5-isothiocynate (FITC) differing in its N- and C-termini. We conducted cellular toxicity and uptake experiments in U-937 and HeLa cells to analyze biocompatibility profiles and translocation efficiencies of Rath peptide with FITC serving as both a cargo and a fluorescent marker. We found that the conjugation of FITC on Rath peptide at N- terminus (FITC-Rath) led to more rapid cellular uptake in U-937 cells and significantly higher cellular uptake in HeLa cells than that which occurred at C-terminus. From DNA microarray analysis, FITC-Rath induced gene expression changes in both U-937 and HeLa cells. Five overlapping regulated genes were identified, and this overlap indicated that FITC-Rath displayed some degree of generality regarding gene responses in the two cell lines used. A real-time quantitative reverse transcriptase-polymerase chain reaction was used to confirm which regulated genes were affected by FITC-Rath. Cell communication, signal transduction, cell surface receptor signaling pathway, signal transducer activity and cellular process, were identified as overlapping biological themes. These data provide useful information on molecular mechanisms for using Rath-based delivery systems.