Objectives: To determine the prevalence of vitamin D deficiency on dried blood spots (DBS) obtained at newborn bloodspot screening (NBS) and thereby test the efficacy of the UK national antenatal supplementation programme in an increasingly ethnically diverse English population. To evaluate the seasonal and ethnic variation in neonatal plasma 25 hydoxyvitamin D (25OHD) and its determinants. Design: Three thousand random DBS samples received at a single regional newborn screening laboratory (52° N) over two one-week periods, one in winter (February 2019) and one in summer (August 2019), were collected. Data was collected from NBS cards on birth weight, gestational age, maternal age, ethnicity, and post code which was replaced with index of multiple deprivation (IMD). 25OHD concentrations were measured on 6mm sub-punch from DBS using quantitative liquid chromatography tandem mass spectrometry adjusted to equivalent plasma values. 25OHD variation with season was assessed using Mann-Whitney U test and ethnic groups compared using Kruskal-Wallis test. Linear regression was used to assess the determinants of 25OHD concentrations. Results: 25OHD measurements were available in 2999 (1580 males) subjects [1499 winter-born and 1500 summer-born]. The majority were white British (59.1%) and born at term (mean SD gestational age of 38.81.8 weeks) with a mean (SD) birth weight of 3306 (565) grams. The overall prevalence of vitamin D deficiency [25OHD<30 nmol/L (12 µg/L)] was 35.7% (n=1070) and insufficiency [30-50 nmol/L (12-20 µg/L)] 33.7% (n=1010). The median (IQR) 25OHD concentration was significantly lower in the winter-born compared to summer-born [29.1 (19.8, 40.6) vs 49.2 (34.3, 64.8) nmol/L respectively; p<0.001]. Across both seasons, when compared to white British babies (41.6 nmol/L), the median 25OHD concentrations were significantly lower in babies of black (30.3 nmol/L; p<0.001), Asian (31.3 nmol/L; p<0.001), any other mixed (32.9 nmol/L; p<0.001), mixed white and black (33.7 nmol/L; p<0.05) and any other white (37.7 nmol/L; p<0.05) ethnicity. The proportion of deficiency was also higher in babies of Asian (48%), black (47%) and mixed ethnicity (38-44%) compared to any other white (34%) or white British (30%) ethnicity. Season of birth, ethnicity, gestation and maternal age accounted for almost 24% of the variation in 25OHD concentrations. Conclusion: The current UK antenatal supplementation programme fails to protect newborns from vitamin D deficiency, especially those from minority ethnic groups who are at high risk of vitamin D deficiency. Nearly 70% of all newborns and 85% of winter-borns had 25OHD concentrations below 50 nmol/L (20 µg/L). Almost 50% of babies of Black or Asian origin were deficient at birth, which explains their high risk of hypocalcaemic complications and rickets if left unsupplemented. Our findings call for an immediate review of the delivery of antenatal and infant vitamin D supplementation programmes and implementation of food fortification in the long term.