Cancer cell promotion of glycolysis provides a promising therapeutic target for cancer treatment. Dihydroartemisinin (DHA) displays cytotoxicity to multiple human tumor cells. However, its effects on pancreatic cancer cells are not well studied. The objective of this study was to investigate the effect of DHA on glucose metabolism and cell viability in JF-305 pancreatic cancer cells. To achieve these goals, cell viability was measured with MTT assay, and the occurrence of apoptosis was detected. Glucose uptake, lactate production, and ATP content were measured. Western blotting was used for the detection of apoptosis-related protein expression. The result showed that DHA caused significant reduction in JF-305 cell viability, arrested the cell phase in G₂/M, induced apoptosis, and decreased the mitochondrial membrane potential and accumulated ROS. DHA also inhibited glucose uptake, lactate generation, and ATP production. Western blotting showed that treatment with DHA increased the activity of caspase-9 and caspase-3, downregulated Bcl-2 expression, and upregulated the expression levels of Bax and Cyto C. Meanwhile, DHA downregulated the Akt/mTOR signaling pathway and inhibited glucose transporter 1 expression. Our data suggest that DHA treatment increased the apoptosis of JF-305 pancreatic cancer cells, and the effect of apoptosis may be associated with the inhibition of glycolysis.