Background: Possible synergism between female sex hormones and vitamin D on periodontitis pathology has not been assessed. Here, the authors investigate effects of estrogen, progesterone, and vitamin D on periodontitis in a population‐based sample and use cell studies to explore mechanistic explanations of the population‐based findings.Methods: The epidemiologic analysis uses cross‐sectional data from the continuous National Health and Nutrition Examination Survey 2001 to 2004. The cross sections include 1,230 women aged 40 to 85 years who received a periodontal examination, responded to questions regarding hormone replacement therapy (HRT), and provided a blood sample for serum vitamin D assessments. For mechanistic cell culture studies, human monocytes were cultured with or without lipopolysaccharide (LPS), estradiol, progesterone, and/or 1,25‐dihydroxyvitamin D3; and transcriptional activity of interleukin (IL)‐6, IL‐1β, B lymphocyte chemoattractant (BLC), and regulated on activation normal T‐cell expressed and secreted (RANTES) was assessed.Results: HRT use (versus none) was associated with higher attachment levels and more teeth only among participants who were vitamin D sufficient (>20 ng/mL). The odds ratio for having moderate/severe periodontitis among users of HRT versus participants who did not use HRT was 0.69 among participants who were vitamin D sufficient and 1.19 in participants who were vitamin D deficient. LPS‐induced IL‐6, IL‐1β, and BLC expression was attenuated in human monocytes treated with estrogen and progesterone. Downregulation of IL‐6 expression by estrogen and progesterone was potentiated when vitamin D was included. LPS‐induced IL‐6 and RANTES expression was decreased, and BLC expression was totally reversed, by vitamin D treatment.Conclusions: The association between HRT and clinical periodontal measures was strongest among women with high vitamin D levels. This association is plausibly mediated via an anti‐inflammatory transcriptional mechanism.