PDAC cells perceive and respond to mechanical stimuli in their extracellular microenvironments (ECMs), playing a crucial role in chemoresistance, while the underlying mechanisms are not fully understood. The progression of various solid tumors is accompanied by metabolic reprogramming. RNA-seq and untargeted metabolomics analysis indicated that stiff substrate may regulate lipid metabolism. The expression of lipogenesis-related genes, including fatty acid synthase (FASN), ATP citrate lyase (ACLY) and acetyl-CoA carboxylase (ACC) was elevated, also the sum of lipid droplets and the triglyceride content. Herein, whether lipid metabolism is involved in matrix stiffness-mediated PDAC chemoresistance and the in-depth mechanism were further explored. Rescue with C75 (FASN inhibitor) validated that fatty acid synthesis participated in matrix stiffness-regulated chemoresistance. Simultaneously, the SCD1 expression was reinforced, consistent with PDAC tissues. The concurrent restraint SCD1 (with inhibitor CAY10566 or shSCD1) and addition of oleic acid confirmed that SCD1 is involved in matrix stiffness-mediated chemoresistance through fatty acid synthesis. In addition, Piezo1 regulated SCD1 expression through the augmentation of Ca(2+) influx, and the PI3K/Akt pathway participated in this process. Taken together, our research sheds light on lipid metabolism exerts an essential role during matrix stiffness-mediated chemoresistance through Piezo1-elicited elevation of SCD1. Our findings delivered a supplement PDAC chemoresistance mechanism mediated by matrix stiffness from the perspective of lipid metabolic reprogramming, and provided a novel strategy for improving clinical therapies.