Liver kinase B1 (LKB1) is a well-known tumor suppressor gene in a variety of human cancers, including breast cancer. However, its role in gemcitabine resistance is unclear. Since gemcitabine in combination with other chemotherapeutic reagents is the first‐line treatment in advanced breast cancer, the aim of the present study was to determine the effect of ectopic expression of LKB1 on chemosensitivity to gemcitabine in the breast cancer MDA-MB-231 cell line. Increasing the expression of LKB1 was found to directly correlate with gemcitabine chemoresistance. Although LKB1 suppressed the cell proliferation rate and clonogenicity in the absence of gemcitabine, it increased the median inhibitory concentration of gemcitabine and clonogenicity of cells in the presence of gemcitabine. Mechanistic analysis indicated that LKB1 was able to protect cells from DNA damage caused by gemcitabine. Furthermore, it was found that LKB1 induced a significant upregulation of cytidine deaminase expression, an important enzyme that accelerates gemcitabine catabolization. Overall, dual characteristics of LKB1 were identified: Suppressing cell growth in normal conditions and enhancing chemoresisitance to gemcitabine, possibly by accelerating degradation of gemcitabine, and protecting cells from DNA damage caused by gemcitabine.