The deficiency of the vitamin D is an important global problem, particularly in children. However, in relation to preschool children from Southern Europe there is a lack of information regarding this issue. The aim of this study was to establish the prevalence of vitamin D deficiency and the association in relation to gender, time spent out doors, physical activity (PA), body mass indeks (BMI), waist and height ratio (WhtR), Mediterranean diet (predictors) and vitamin D status (outcome) in healthy preschool children. The participants were preschool children (all aged 5 to 6 years) from South Croatia. All participants were tested during mandatory medical examination, 6-7 months prior to school enrollment. The PA was calculated using Pre-Paq questionnaire categorizing PA in to five levels (from sedentiary to high level of physical activity). The prevalence of vitamin D deficiency was high; 58% of children had vitamin D, 25(OH)D levels <50 nmol/l (VDD), while in the remaining 29% an insufficient concentration of vitamin D, 25(OH)D 50-75 nmol/l (VDI) was reported. The boys had higher levels of vitamin D than girls (49.61±19.67 i 43.37±20.23 nmol/l, respectively; ttest: 2.52, P < 0.01). Using multinomial regression in order to calculate the effectof vitamin D and the sufficient level (25(OH)D >75 nmol/l) as the reference value, the gender was determined as a significant predictor for vitamin D status, with boys being at lower risk for vitamin D deficiency in relation to girls (OR: 0.33; 95%CI: 0.15-0.74). As a significant factor of high vitamin D levels, the KIDMED index higher than 7 points was indicated, which suggested an optimal following of the Mediterranean diet. The implementation of Bayesian model selection enable devidence of the postive correlation between vitamin D and the intake of more than two servings of fruit per day as well as the olive oil consumption, where as skipping meals had negative effect on vitamin D levels. The model could have accounted for only 5.2% of variations in blood levels of VD, but it was statistically significant. These results indicated the high prevalence of the vitamin D deficiency in the preschool children from South Croatia, which is especially alarming given the geographical position of the studied region (42 o N) as well as the high number of sunlight hours(>2600 hours per year). Further investigation for other potential correlations with vitamin D is essential, also the establishment of a public health platform for vitamin D supplementation.