Objective <p>To explore the effects of icaritin on pancreatic cancer cell proliferation and the mechanisms underlying cell death.</p> Methods <p>Transcriptomic and lipidomic analyses were performed on the pancreatic cancer cell lines pancreatic carcinoma 1 (PANC-1) and ascites of the pancreas carcinoma 1 (ASPC1) treated with icaritin (0, 25 µ, mol/L) to profile global gene expression and lipid metabolism alterations. Key cholesterol biosynthesis genes were validated via quantitative reverse transcription polymerase chain reaction and Western blot. Additionally, cell viability was assessed using luminescent assays, while cytoplasmic vacuolization (paraptosis marker) was observed microscopically. Total cholesterol levels were quantified enzymatically, and lipid species (e.g., phosphatidylcholine, triglycerides) were analyzed by principal component analysis and pathway enrichment.</p> Results <p>Icaritin significantly altered lipid metabolism in pancreatic cancer cells by elevating membrane lipids such as phosphatidylcholine, ceramide, sphingomyelin, and phosphatidylethanolamines (<i>P</i>&lt;0.05). Concurrently, it reduced the levels of energy-supplying lipids including triglycerides, diglycerides, and acylcarnitines (<i>P</i>&lt;0.05). There was also a notable decrease in cholesteryl ester 24:1 levels, which is consistent with the suppression of cholesterol biosynthesis (<i>P</i>&lt;0.05 or <i>P</i>&lt;0.01). Icaritin inhibited the proliferation of PANC-1 and ASPC1 cells by downregulating key cholesterol biosynthesis genes, such as 3-hydroxy-3-methylglutaryl coenzyme A reductase and squalene epoxidase genes (<i>P</i>&lt;0.01).</p> Conclusions <p>Icaritin disrupts lipid metabolism and inhibits cholesterol biosynthesis in pancreatic cancer cells, leading to non-apoptotic cell death. This novel mechanism of action provides new therapeutic possibilities for the treatment of pancreatic cancer and highlights its potential as a targeted anticancer agent.</p>