Matrix effects are a known problem with immunoassays measuring serum 25-hydroxyvitamin D 25(OH)D. To determine if the inverse association between serum 25(OH)D and serum cholesterol concentrations is a function of assay method: Diasorin Liaison 25(OH) Vitamin D Total Assay (Liaison Total Assay), an immunoassay, compared with liquid chromatography tandem mass spectrometry (LC-MS/MS). Canadian Health Measures Survey data and biobank serum (White males aged 20–79 y, n = 392) were evaluated for bias in serum 25(OH)D using Bland-Altman plots. Differences in serum 25(OH)D (Liaison Total Assay − LC-MS/MS) were compared among non-HDL-cholesterol <4.2 (n = 295) compared with ≥4.2 (n = 97) mmol/L and total cholesterol groups <5.2 (n = 256) compared with ≥5.2 (n = 136) mmol/L, and associations tested between 25(OH)D and non-HDL-cholesterol or total cholesterol concentrations, using regression. Serum 25(OH)D measured using Liaison Total Assay ranged from 10.7 to 137.0 nmol/L and 14.4 to 137.9 nmol/L by LC-MS/MS. Liaison Total Assay − LC-MS/MS showed a negative bias of 5.5 (95% limits of agreement −23.8, 12.7) nmol/L. Differences in 25(OH)D were −4.0 ± 9.0 (±SD) nmol/L if non-HDL-cholesterol was <4.2 mmol/L and −10.2 ± 8.7 nmol/L if ≥4.2 mmol/L (P < 0.0001). Differences in 25(OH)D, if total cholesterol was <5.2 mmol/L, were −3.4 ± 8.6 nmol/L and −9.6 ± 9.3 nmol/L if ≥5.2 mmol/L (P < 0.0001). Serum non-HDL-cholesterol (beta −3.17, P = 0.0014) and total cholesterol (beta −2.77, P = 0.0046) were inversely associated with Liaison Total Assay 25(OH)D (adjusted for age, fasting, and body mass index), but not with LC-MS/MS measured 25(OH)D. Interference by these lipoproteins was not eliminated by standardization of the Liaison Total Assay. Similar associations were observed with triglycerides as for the lipoproteins. Total cholesterol inversely associates with 25(OH)D, which is likely due to elevated non-HDL-cholesterol lipoprotein or triglyceride interference with the Liaison Total Assay. This is important as elevated cholesterol is common, and an underestimation of vitamin D status could be an unnecessary cause for concern.