Cheese intake has been shown to decrease total cholesterol and LDL cholesterol concentrations when compared to butter of equal fat content. An untargeted metabolite profiling may reveal exposure markers of cheese but may also contribute with markers which can help explain how the intake of cheese affects cholesterol concentrations. Twenty-three subjects collected 2 × 24 h urine samples after 6 weeks of cheese and 6 weeks of butter intake with equal amounts of fat in a cross-over intervention study. The samples were analyzed by UPLC-QTOF/MS. A two-step univariate data analysis approach using linear mixed model was applied separately for positive and negative ionization mode: In the first step a total of 44 features related to treatment were identified and in the second step 36 of these features were related to total cholesterol concentrations. Cheese intake resulted in increased urinary indoxyl sulfate, xanthurenic acid, tyramine sulfate, 4-hydroxyphenylacetic acid, isovalerylglutamic acid and several acylglycines including isovalerylglycine, tiglylglycine and isobutyrylglycine when compared to butter intake of equal fat content. The biological mechanisms of action linking the metabolites to cholesterol concentrations need to be further explored.