Gemcitabine (GEM) is the front-line standard chemotherapy used for the treatment of pancreatic cancer; however, chemoresistance to GEM remains the major obstacle to the successful control of this disease. Both the expression levels and activity of aldehyde dehydrogenase 1A1 (ALDH1A1) are important features of tumor-initiating and/or cancer stem cell properties in multiple types of human cancer. As one of the intrinsic properties of cancer stem cells is drug resistance, in this study, we examined the correlation between the level and activity of endogenous ALDH1A1 and GEM resistance in the MIA PaCa-2 cell line that contains high expression levels and activity of ALDH1A1. We used small interfering RNAs (siRNAs) to deplete ALDH1A1 and investigate its potential role in conferring GEM resistance. The ALDH1A1 knockdown markedly reduced ALDH1A1 expression and activity and inhibited cell proliferation. Moreover, the combination of ALDH1A1-siRNA and GEM significantly decreased cell viability, increased apoptotic cell death and increased the accumulation of cells at the S-phase compared to the controls. Our data also demonstrated that ALDH1A1 expression and activity were significantly higher in the GEM-resistant MIA PaCa-2 cell line (MIA PaCa-2/GR), compared to the parental MIA PaCa-2 cell line (MIA PaCa-2/P). In the MIA PaCa-2/GR cells, the combination of ALDH1A1-siRNA and GEM also showed a significant decrease in cell viability and an increase in apoptotic cell death, emphasizing the importance of ALDH1A1 in both intrinsic and acquired GEM resistance. This potentially powerful combination treatment of ALDH1A1-siRNA and GEM warrants further investigation as an effective therapeutic regimen to overcome the resistance of pancreatic cancer to GEM.