Pancreatic cancer is the seventh leading cause of cancer-related death worldwide and is known as “the king of cancers”. Currently, gemcitabine (GEM) as the clinical drug of choice for chemotherapy of advanced pancreatic cancer has poor drug sensitivity and ineffective chemotherapy. Nardoguaianone L (G-6) is a novel guaiane-type sesquiterpenoid isolated from Nardostachys jatamansi DC., and it exhibits anti-tumor activity. Based on the newly discovered G-6 with anti-pancreatic cancer activity in our laboratory, this paper aimed to evaluate the potential value of the combination of G-6 and GEM in SW1990 cells, including cell viability, cell apoptosis, colony assay and tandem mass tags (TMT) marker-based proteomic technology. These results showed that G-6 combined with GEM significantly inhibited cell viability, and the effect was more obvious than that with single drug. In addition, the use of TMT marker-based proteomic technology demonstrated that the AGE-RAGE signaling pathway was activated after medication-combination. Furthermore, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) assays were used to validate the proteomic results. Finally, apoptosis was detected by flow cytometry. In conclusion, G-6 combined with GEM induced an increase in ROS level and a decrease in MMP in SW1990 cells through the AGE-RAGE signaling pathway, ultimately leading to apoptosis. G-6 improved the effect of GEM chemotherapy and may be used as a potential combination therapy for pancreatic cancer.