Abstract Purpose Many dietary factors have either proinflammatory or anti-inflammatory properties. We previously developed a dietary inflammatory index (DII) to assess the inflammatory potential of diet. In this study, we conducted a construct validation of the DII based on data from a food frequency questionnaire and three inflammatory biomarkers in a subsample of 2567 postmenopausal women in the Women's Health Initiative Observational Study. Methods We used multiple linear and logistic regression models, controlling for potential confounders, to test whether baseline DII predicted concentrations of interleukin-6, high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor alpha receptor 2, or an overall biomarker score combining all three inflammatory biomarkers. Results The DII was associated with the four biomarkers with beta estimates (95% confidence interval) comparing the highest with lowest DII quintiles as follows: interleukin-6: 1.26 (1.15–1.38), Ptrend < .0001; tumor necrosis factor alpha receptor 2: 81.43 (19.15–143.71), Ptrend  = .004; dichotomized hs-CRP (odds ratio for higher vs. lower hs-CRP): 1.30 (0.97–1.67), Ptrend  = .34; and the combined inflammatory biomarker score: 0.26 (0.12–0.40), Ptrend  = .0001. Conclusions The DII was significantly associated with inflammatory biomarkers. Construct validity of the DII indicates its utility for assessing the inflammatory potential of diet and for expanding its use to include associations with common chronic diseases in future studies.