Multidrug resistance (MDR) is a major problem in cancer chemotherapy. It was previously reported that a red ginseng saponin, 20(S)-ginsenoside $Rg_3$ could modulate MDR in vitro and extend the survival of mice implanted with ADR-resistant murine leukemia P388 cells. This study examined the cytotoxicity of $Rg_3$ on normal and transformed cells, along with its effect on the membrane fluidity. The cytotoxicity study revealed that 120 ${mu}M;of;Rg_3$ was cytotoxic against a multidrug-resistant human fibroblast carcinoma cell line, KB V20C, but not against normal WI 38 cells in vitro. Flow cytometric analysis using rhodamine 123 as the artificial substrate showed that $Rg_3$ promoted the accumulation of rhodamine 123 in ADR-resistant murine leukemia P388 cells in vivo. Fluorescence polarization studies using the hydrophilic fluorescent probe, DPH, and hydrophobic probe, TMA-DPH, showed that 20 ${mu}M; Rg_3$ induced a significant increase in fluorescence anisotropy in KB V20C cells but not in the parental KB cells. These results clearly show that $Rg_3$ decreases the membrane fluidity thereby blocking drug efflux.