Free thiol groups of intra and extracellular molecules are considered to be antioxidative and to protect cells from damage caused by free radicals. However, the associations of serum total thiol levels (TTL) with the incidences of the four most frequent cancer sites have not yet been investigated in a large population-based, prospective study. TTL was measured in case-cohort design in a sample from the population-based, Norwegian Tromsø 3 study (cancer cases: n = 941; random subcohort: n = 1,000) and was repeatedly measured at Tromsø 5. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) were estimated by weighted multivariable-adjusted Cox regression with time-dependent modeling of TTL for incident lung, colorectal, breast and prostate cancer. High serum TTL were associated with a reduced risk of all four major cancers. The associations with lung (top vs. bottom tertile: HR, 0.64; 95% CI, 0.41, 0.99) and breast cancer (top vs. bottom tertile: HR, 0.64; 95% CI, 0.42, 0.96) were statistically significant, whereas associations with colorectal (top vs. bottom tertile: HR, 0.79; 95% CI, 0.54, 1.16) and prostate cancer (top vs. bottom tertile: HR, 0.79; 95% CI, 0.53, 1.17) were not statistically significant but pointed in the same protective direction. These findings from a large, prospective Norwegian cohort study suggest a preventive role of thiols against the development of the four most frequent cancers. Whereas associations with breast and lung cancer could be shown with statistical significance, larger studies are needed to corroborate potential associations of TTL with colorectal and prostate cancer.