Cell-penetrating peptide (CPP) can be used in pharmaceutics as a highly efficient drug delivery transporter.  In this study, four tumor cell lines (MCF-7, MDA-MB-231, C6, and B16F10) were used to observe the uptake of fluorescein isothiocyanate (FITC) labeled CPP and the effects of time and concentration of CPP  on cell penetration was studied.  The CPP exocytosis on C6 cell line was observed, and its exocytosis kinetics was described by zero order equation.  In addition, low-temperature condition (4 ℃) and endocytosis inhibitors were utilized to investigate the mechanism of CPP uptake by cells.  Low-temperature condition did not show significantly inhibition on CPP uptake.  Heparin, a membrane glycoprotein receptor inhibitor, showed strong  inhibition effect (only 3%−10% of the control) on CPP uptake.  Chlorpromazine, chloroquine and 5-(N-ethyl-N- isopropyl)-amiloride (EIPA) showed little effect on CPP uptake.  This study indicated that CPP penetration had little selectivity on cell type, but the amount and rate of CPP penetration into cells were related to the type of cell lines.  The adsorption of CPP on cell membrane induced by sulfate proteoglycan plays an important role on CPP penetration.