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Vitamin D (VD) serum levels, and keratinocytic basal expression of vitamin D receptor (VDR) before treatment of actinic keratoses (AK) have been previously reported as possible biomarkers of the response of AK to treatments. We intended to evaluate the association between these and other serum and immunohistochemical parameters with the response of AK to treatment with topical ingenol mebutate (IM). Twenty-five patients with AK on the head were treated with topical IM 0.015% gel once daily for 3 days. Biopsies were taken at baseline and 6 weeks after treatment. Immunohistochemical staining was performed for VDR, P53, Ki67, Aurora B, Survivin and β-catenin. Basal serum 25(OH)D levels were determined. IM was more effective for KIN I and II AKs than in KIN III, and histological responders showed significantly higher serum VD levels (30.278 [SD 8.839] ng/mL) than nonresponders (21.14 [SD 7.079] ng/mL, p = 0.023). In addition, mean basal expression of VDR (45.63 [SD 16.105] %) increased significantly (57.92 [SD 14.738] %, p = 0.003) after treatment with IM. A significant decrease after treatment in the expression of several markers of aggressiveness and progression to squamous cell carcinoma, namely P53, Ki-67, aurora B kinase and survivin, was also observed. Our results support a relationship between VD status and the response of AK to treatment with topical IM, suggesting that its previous correction to proper serum levels in VD-deficient patients could improve the response of AK to the treatment.