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In view of research suggesting a possible beneficial impact of vitamin D on systemic inflammatory response, the authors decided to investigate an influence of vitamin D supplementation on serum levels of certain inflammatory markers in obese patients. The current study included such biomarkers as interleukin-6 (IL-6), pituitary adenylate cyclase-activating peptide (PACAP), advanced oxidation protein products (AOPP), C-X3-C Motif Chemokine Ligand 1 (CX3CL1), monocyte chemoattractant protein-1 (MCP-1), and nitric oxide (NO). The measurements were performed with the ELISA method before and after 3-month-long supplementation of 2000 IU of vitamin D orally. The results showed that the therapy did not induce any statistically significant changes in serum levels of MCP-1, IL-6, CX3CL1, and PACAP. The supplementation was related to a significant increase in measurements of NO and AOPP levels, although the correlation analysis between vitamin D concentration after its supplementation and the concentration of the molecular parameters did not show significant relation. In conclusion, our study seems to contradict certain aspects of findings available in the literature regarding the vitamin D’s impact.