Background Subjects with atopic dermatitis (AD) have defects in antimicrobial peptide (AMP) production possibly contributing to an increased risk of infections. In laboratory models, vitamin D can alter innate immunity by increasing AMP production. Objective To determine if AD severity correlates with baseline vitamin D levels, and to test whether supplementation with oral vitamin D alters AMP production in AD skin. Methods This was a multi‐centre, placebo‐controlled, double‐blind study in 30 subjects with AD, 30 non‐atopic subjects, and 16 subjects with psoriasis. Subjects were randomized to receive either 4000 IU of cholecalciferol or placebo for 21 days. At baseline and day 21, levels of 25‐hydroxyvitamin D (25OHD), cathelicidin, HBD‐3, IL‐13, and Eczema Area and Severity Index (EASI) and Rajka‐Langeland scores were obtained. Results At baseline, 20% of AD subjects had serum 25OHD below 20 ng/mL. Low serum 25OHD correlated with increased Fitzpatrick Skin Type and elevated BMI, but not AD severity. After 21 days of oral cholecalciferol, mean serum 25OHD increased, but there was no significant change in skin cathelicidin, HBD‐3, IL‐13 or EASI scores. Conclusions This study illustrated that darker skin types and elevated BMI are important risk factors for vitamin D deficiency in subjects with AD, and highlighted the possibility that seasonality and locale may be potent contributors to cathelicidin induction through their effect on steady state 25OHD levels. Given the molecular links between vitamin D and immune function, further study of vitamin D supplementation in subjects with AD is warranted.