Multidrug resistance (MDR) of tumor cells attenuates the efficacy of anticancer drugs and has become the main reason for chemotherapy failure. It is indispensable to establish an effective way to reverse multidrug resistance. Our previous work has shown that downregulation of the ERK/MAPK signaling pathway activity can reverse the drug resistance of resistant cells. Furthermore, the effect of signal transduction is strongly associated with lipid rafts. The drug resistance is reversed successfully after lipid rafts are destroyed by heptakis(2,6-di-O-methyl)-β-cyclodextrin (MβCD). However, reversal of the drug resistance is not associated with downregulation of ERK1/2 expression. Cell membrane permeability may increase when lipid rafts are destroyed by MβCD, causing the reversal of drug resistance due to an increased drug accumulation in cytoplasm. To minimize the influence of MβCD on the cell membrane structure, we selected flotillin, a marker protein of lipid rafts, as the target molecule to further investigate the mechanism of changes in drug resistance after destruction of lipid rafts. The effect of flotillin on the reversal of drug resistance was examined using RNA interference (RNAi) in a retroviral system in human drug resistant strains of colorectal cancer cell line HCT-15. The results demonstrate that flotillin-1 downregulation by RNAi (Flot1-RNAi) reduced the drug resistance, caused cell cycle arrest and decreased the expression of ERK1/2; however, apoptosis was not significantly affected. Knockdown of flotillin-2 by RNAi (Flot2-RNAi) had effects similar to those of Flot1-RNAi except that the effects on ERK1/2 expression and apoptosis were different. Screening of multiple pathways indicated that the PI3K/Akt signaling pathway was closely related. This experiment demonstrates an association between PI3K and drug resistance through the activation of PI3K and suggests that PI3K may play a key role during the development of resistance in CRC. The results reveal that the levels of IRS-1 and PI3K proteins in the Flot1-RNAi and Flot2-RNAi groups were significantly downregulated. Knockdown of flotillins by RNAi reduced the resistance of HCT-15/ADM cells; and the results on Akt pathway indicate a decrease in resistance after lipid raft destruction. These data confirm that knockdown of flotillin reduces the resistance of HCT-15/ADM cells and the mechanism may be relevant to the PI3K/Akt pathway. Additionally, flotillin may be used as a potential target for chemotherapy in the treatment of colorectal cancer.