Rationale: Although it is known that High-fat diet (HFD) promotes the development of pancreatic ductal adenocarcinoma (PDAC), no direct link between HFD and cancer has been identified. Previously, we showed that ATP production by cancer cells depends on fatty acid oxidation (FAO); therefore, we hypothesized that blocking FAO may prevent HFD-induced promotion of PDAC growth. Methods: To determine whether FAO is increased in PDAC patients, we analyzed a tissue microarray by immunohistochemical staining to detect carnitine palmitoyl transferase I. To block FAO, SLC25A20 (carnitine-acylcarnitine carrier) was knocked down in cancer cells, which was implanted for xenograft in mice and treated with a high-fat diet (HFD, 60% fat). To compare cancer development including survival rates, and histopathological differences were analyzed by crossbreeding of KPC mice (Kras(G12D/+;) Trp53(R172H/+;) Pdx1-Cre) with KPC/Slc25a20(+/-) mice. Results: SLC25A20 knockdown in cancer cells reduced ATP production and inhibited cell growth. Proteome analysis revealed that SLC25A20 knockdown reduced cancer cell growth significantly due to inactivation of mTOR via decreased ATP production, ultimately leading to cell death. The median survival time of KPC/Slc25a20(+/-) tumor-bearing mice was 3.1 weeks longer than that of KPC tumor-bearing mice. In mice fed an HFD, the growth of xenografts derived from SLC25A20 knockdown PDAC cells was 65-95% lower than that of xenografts derived from control cells. Conclusion: Blocking FAO by SLC25A20 knockdown reversed HFD-induced promotion of PDAC growth.