Background: We aimed to examine if 25-hydroxyvitamin D (25(OH)D) was related to the peripheral immunological and inflammatory signature both at birth, and in newly diagnosed patients with childhood type 1 diabetes (T1D) and their healthy controls; (2) Methods: The birth cohort consisted of 470 patients and 500 healthy controls. Dried blood samples were collected from the neonates in the period 1981–1999. The newly diagnosed cohort consisted of 460 patients and 453 siblings. Serum samples were collected in the period 1997–2005. A variety of peripheral immune mediators were measured and compared to total 25(OH)D levels (25(OH)D2 + 25(OH)D3). For each immune mediator, the relative change (RC) in the mean level was modeled by robust log-normal regression and correction for multiple testing was performed; (3) Results: Two associations were identified; there was a negative association between 25(OH)D (10 nmol/L increase) and leptin (RC (95% confidence interval (CI)), 0.98 (0.96; 1.00)), and a positive association between 25(OH)D (10 nmol/L increase) and the chemokine, chemokine (c-x-c motif) ligand (CXCL) 8 (RC (95% CI), 1.07 (1.01; 1.13)); (4) Conclusion: CXCL8 and leptin have significant associations with levels of 25(OH)D in the newly diagnosed cohort. These results do not indicate a strong influence of 25(OH)D on the peripheral immunological or inflammatory signature.