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Background: Vitamin D3 deficiency, which is associated with several other diseases such as high blood sugar levels, insulin resistance, and the risk of developing metabolic syndrome (MetS) is prevalent all over the world. Objectives: This study aimed to determine the prevalence of vitamin D3 deficiency and MetS and evaluate their association in patients referred to the central laboratory of Academic Center for Education, Culture, and Research (ACECR) in Mashahd, Iran. Methods: This cross-sectional study was conducted on 1,214 patients aged 15 - 75 years referred to the central laboratory of ACECR in Mashhad, Iran, in 2018. Participants with MetS were identified using the Adult Treatment Panel III (ATPIII) criteria. Biochemical parameters and vitamin D3 levels were assessed using the Mindray instrument and the high-performance liquid chromatography (HPLC) method, respectively. The subjects were divided into four groups in terms of serum vitamin D3 concentration as follows: Deficient (< 10 ng/mL), insufficient (10 - 30 ng/mL), sufficient (30 - 80 ng/mL), and toxic (80 < ng/mL). Results: From a total of 1,214 subjects, 15.0% had vitamin D3 deficiency, 53.2% showed insufficiency, 31.1% were normal with sufficient vitamin D3, and 0.7% suffered from vitamin D3 toxicity. Therefore, 437 (49.7%) females and 209 (62.6%) males suffered from vitamin D3 insufficiency. Overall, 27.6% of females and 33.2% of males had MetS (P < 0.05). Subjects with and without MetS showed no significant difference in serum levels of vitamin D3 (P = 0.608). In addition, there was a significant direct correlation between vitamin D3 levels and systolic blood pressure (SBP) in the group with MetS. Conclusions: A high proportion of subjects had MetS and insufficient vitamin D3 levels. There were no significant differences between the serum vitamin D3 levels in participants with and without MetS. [ABSTRACT FROM AUTHOR]