The aim of this study was to develop a valid and reliable questionnaire to assess vitamin D-related knowledge, attitude and practices in Tehranian adults, who may be at increased risk of vitamin D deficiency. This study was conducted on 572 individuals, aged ≥20 years from public health care centers in Tehran, Iran. Based on results of a literature review and in-depth interviews, the 38-item vitamin D-related KAP questionnaire (D-KAP-38) with four subscales was developed: (1) general knowledge; (2) nutritional knowledge; (3) attitudes; (4) behaviors. Validity of the D-KAP-38 questionnaire was assessed, utilizing face, content, and construct validity methods. Internal consistency was calculated to assess reliability of the current developed questionnaire. A total of 572 (54.1% female) adults, aged 30.2 ± 7.9 years, participated in the study. All items were perceived as relevant and comprehendible by participants. Content validity was confirmed by a panel of experts. The internal consistency, as measured by Cronbach’s alpha coefficients, exceeded the minimum reliability standard of 0.60 for four subscales. Exploratory factor analysis suggested a four-factor construct and the results of the confirmatory factor analysis indicated acceptable fit indices for the proposed model. No ceiling effects were observed except for general knowledge (1.2%). Floor effects detected were 0%, 1.1%, 2.4%, and 8.7% for practice, attitude, general knowledge, and nutrition knowledge, respectively. General knowledge had the highest score (79.59 ± 14.52) and nutrition knowledge had the lowest (42.58 ± 20.40), among the four sub-scales. Results confirm the initial validity and reliability of D-KAP-38 questionnaire. Further investigations in different populations are recommended.