Fibroblasts, keratinocytes, mast cells, and other cells participate in hypertrophic scar formation and express the vitamin D receptor. We investigated the association between vitamin D deficiency and the biomechanical properties of hypertrophic burn scars. This cross-sectional study analyzed 486 participants enrolled from May 1, 2013 to April 30, 2017. When complete wound healing was agreed with by the two opinions, blood sampling and scar evaluation were performed. The values of melanin and erythema, trans-epidermal water loss (TEWL), and scar distensibility and elasticity were measured using pigment- and TEWL-measuring devices and a suction skin elasticity meter. 25(OH) vitamin D deficiency was defined as plasma level of <20 ng/ml. The vitamin D-deficient patients had significantly higher mean values of scar melanin and TEWL (P = .032, P = .007), whereas scar erythema level was similar. They also showed significantly lower values of Uf (final distensibility; P < .001), Ua/Uf (gross elasticity; P < .001) and Ur/Uf (biological elasticity; P = .014), and higher value of Uv/Ue (viscoelasticity or potency against interstitial fluid shift; P = .016). In multiple linear regression analysis, Uf, Ua/Uf, Uv/Ue, and Ur/Uf were significantly affected by 25(OH)-vitamin D level in deficient patients (Uf, P = .017; Ua/Uf, P = .045; Uv/Ue, P = .024; Ur/Uf, P = .021). Our results demonstrated that vitamin D deficiency was significantly related to increased pigmentation, decreased skin barrier function, low scar distensibility and elasticity, and slow interstitial fluid movement in burn patients.