Abstract Background: Pancreatic cancer is the third leading cause of cancer-related death in the United States. Total serum cholesterol (TSC) may predict cancer risk, although its role independent of statins remains elusive. We examined the association between TSC and pancreatic cancer risk independent of statins. Methods: A nested case–control analysis was conducted among statin-naïve patients within The Health Improvement Network (THIN), a United Kingdom–based general practice database. Cases were >40 years old and diagnosed with pancreatic cancer after at least 6 months of follow-up. Controls were selected by incidence density sampling and matched by age, sex, practice site, and follow-up. Primary exposure was TSC (mmol/L) prior to index date. Conditional logistic regression estimated ORs for pancreatic cancer risk associated with TSC. Sensitivity analyses were conducted among nondiabetics. Results: Among 1,241 cases and 3,307 matched controls, an average 8% reduction was observed in pancreatic cancer risk per mmol/L increase in TSC [OR 0.92, 95% confidence interval (CI): 0.85–1.00; nondiabetics: OR 0.91, 95% CI: 0.83–0.99]. When TSC was measured at 12-month intervals prior to diagnosis, the OR between TSC and pancreatic cancer was 0.88 at 0 to 12 months (95% CI: 0.77–1.00; nondiabetics: OR 0.81, 95% CI: 0.68–0.96). No significant association was seen at subsequent discrete intervals before index date. Conclusions: TSC is a significant predictor of short-term risk for pancreatic cancer. This risk increase associated with lower TSC was independent of statins. Impact: TSC could serve as a biomarker for risk stratification, screening, and early diagnosis of pancreatic cancer in future clinical prediction models.