Vitamin D deficiency is a globally important problem, particularly in children, but there is a lack of information regarding this deficiency in preschool children from southeastern Europe. This study aimed to establish the levels of 25-hydroxyvitamin D (25(OH)D) and associations of gender, time spent outdoors, physical activity (PA), and body mass index (as predictors) with the 25(OH)D level (outcome) in healthy preschool children. The participants were preschoolers (all 5–6 years of age) from southern Croatia. All the participants were tested during their mandatory medical examination 6–7 months prior to school enrollment. The PA was obtained using the preschool-age physical activity questionnaire (Pre-PAQ), which categorizes PA into five levels (from sedentary to vigorous PA). The prevalence of 25(OH)D deficiency was high: 58% of the children had 25(OH)D levels of <50 nmol/L (deficiency), and an additional 29% had an insufficient level of 25(OH)D (50–75 nmol/L). Boys had higher levels of 25(OH)D than girls. A multinomial regression using 25(OH)D categories as the outcome and a sufficient level (>75 nmol/L) as the reference value identified gender as the only significant predictor of 25(OH)D status, with boys being at lower risk for 25(OH)D deficiency than girls. These results showed a high prevalence of 25(OH)D deficiency in preschoolers from the southern part of Croatia, which is additionally alarming based on the geographical position of the studied region (42° N) and its high number of sunshine hours (>2600 h per year). Future studies examining other potential correlates of 25(OH)D in the region are warranted.