Pancreatic cancer is one of the most lethal types of cancer, due to difficulty in early detection and the limited efficacy of available treatments. Erlotinib is used to inhibit the epidermal growth factor receptor for the treatment of pancreatic cancer; however, erlotinib resistance is a major issue and the mechanisms underlying the development of erlotinib resistance remain unclear. To better understand the alterations in tumor metabolism by acquired resistance to erlotinib, an erlotinib-resistant pancreatic cancer cell line (HPAC-ER) was established, followed by a comparison of the metabolic characteristics between these cells and their erlotinib-sensitive parental cells (HPAC). This comparison was accomplished through mass spectrometry-based targeted metabolic profiling. Five metabolite groups (acylcarnitines, amino acids and biogenic amines, glycerophospholipids, sphingolipids and monosaccharides) were semi-quantified and compared statistically. These results revealed significant differences between the two groups of cells. A significant increase in the level of short-chain acylcarnitines and selected lysophosphatidylcholines, and a significant decrease in the level of acyl-alkyl-phosphatidylcholines and one sphingolipid, were observed in the HPAC-ER cells compared with the HPAC cells. The metabolic changes observed in the present study support the theory that there are increased metabolic demands in erlotinib-resistant cancer, reflecting the changes in acetyl-CoA-associated and choline phospholipid metabolism. These findings will aid in elucidating the changes that occur in pancreatic cancer metabolism through the acquired resistance to erlotinib, and in the identification of biomarkers for the early detection of pancreatic cancer.