In a prospective cohort study, we examined 62 patients undergoing major surgical cancer therapy for Toll-like receptor 4 (TLR4) gene polymorphisms (Asp299Gly and Thr399Ile) and their influence on cytokine levels pre- and postoperatively, as well as cytokine levels after whole blood lipopolysaccharide (LPS) stimulation. Incidence of the TLR4 gene single nucleotide polymorphism (SNP) Asp299Gly/Thr399Ile was 14.5% (9/62). Overall, mortality was unaffected by the TLR4 SNP. Preoperative cytokine levels were low, with most of the values of cytokines being below the detection levels. After preoperative stimulation of whole blood with 50 pg/mL LPS, TNF-alpha and IL-6 values increased significantly in both groups. However, no significant influence was detectable between the TLR4 SNP group and the wild type group (WT group). Postoperative IL-6 levels, but not TNF-alpha levels, were significantly increased in both groups. Postoperative LPS stimulation resulted in significantly lower TNF-alpha levels compared with preoperative induction, with a more than 2.3-fold decrease in the TLR4 SNP group: 310.83 pg/mL (SD: 117.53) to 134.08 pg/mL (SD: 91.49; P < 0.001) and a 2.2-fold decrease in the WT group: 422.97 pg/mL (SD: 662.57) to 191.68 pg/mL (SD:147.26; P = 0.031). IL-6 levels after stimulation were comparably decreased with similarly no significant difference between the two groups. We conclude that the TLR4 polymorphism Asp299Gly/Thr399Ile has no influence on cytokine release after LPS stimulation in the early and late course after major surgery. The LPS adaptation effect of cytokine release after surgery is furthermore not affected by the presence of the TLR4 polymorphism Asp299Gly/Thr399Ile.