The colon possesses a unique physiological environment among human organs, where there is a highly viscous body fluid layer called the mucus layer above colonic epithelial cells. Dysfunction of the mucus layer not only contributes to the occurrence of colorectal cancer (CRC) but also plays an important role in the development of chemoresistance in CRC. Although viscosity is an essential property of the mucus layer, it remains elusive how viscosity affects chemoresistance in colon cancer cells. In this study, the influence of viscosity on their chemoresistance was elucidated by culturing colon cancer cells in media of different viscosities supplemented with doxorubicin (DOX). The viscosity range was adjusted from 99.4 mPa s to 776.6 mPa s by adding polyethylene glycol of different molecular weights in culture medium. Cell viability in the high viscosity medium was higher than that in the low viscosity medium. Expression of chemoresistance-related genes such as ABCC2 and ABCG2 increased when cells were cultured in the high viscosity medium. Furthermore, cell migration increased while proliferation decreased when cells were cultured in the high viscosity medium. The colon cancer cells cultured in the high viscosity medium exhibited high expression of p21 mRNA. The results suggested that viscosity could affect the resistance of colon cancer cells to DOX by regulating the expression of chemoresistance-related and proliferation-related genes.