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Research Question: Is low serum 25-hydroxyvitamin D (25(OH)D) associated with an increased risk of miscarriage in women who presented with threatened miscarriage to the Early Pregnancy Assessment Clinic (EPAC)?, Design: This was a secondary retrospective analysis using archived serum samples from a randomized, double-blind, placebo-controlled trial. Stored serum samples from 371 women presenting to the EPAC with threatened miscarriage during the first trimester were assayed for 25(OH)D by liquid chromatography-mass spectrometry., Results: The overall miscarriage rate was 45/371 (12.1%) in the whole cohort. After grouping vitamin D insufficiency and vitamin D sufficiency together into a 'non-deficient' group and excluding participants who underwent termination of pregnancy, there was no difference in the miscarriage rate between those who were vitamin D deficient compared with those who were not (25/205, 12.2% versus 20/157, 12.7%, P= 0.877, odds ratio 0.951, 95% CI 0.507-1.784). When analysed according to the number of gestational weeks, the miscarriage rate was significantly higher in the vitamin D non-deficient group than the vitamin D-deficient group in women who presented at 6 gestational weeks or earlier (13/33 [39.4%] versus 10/58 [17.2%], P= 0.019), but there were no statistically significant differences between the two groups presenting at later gestations. There was no difference in the vitamin D level in women who had a miscarriage compared with those who had a live birth (48 [37-57] versus 47 [37-58] nmol/l, P= 0.725 median [25th-75th percentile])., Conclusions: A low serum vitamin D concentration was not associated with an increased risk of miscarriage in women with threatened miscarriage presenting to the EPAC., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)