Evaluate associations between preconception 25-hydroxyvitamin D (25(OH)D) and biomarkers in female and male partners on live birth, pregnancy loss, and semen quality. Secondary analysis using the FAZST trial of couples seeking infertility treatment at 4 US centers (2013-2017). A target trial emulation framework was applied to estimate associations. Couples were followed for nine months or through pregnancy. Preconception concentrations of 25(OH)D (primary) and associated biomarkers: vitamin D binding protein (VDBP), calcium, free vitamin D, bioavailable vitamin D. Live birth and pregnancy loss were ascertained via self-report and medical records. Semen quality was ascertained six months after enrollment. Log-binomial regression estimated risk ratios and 95% confidence intervals (CI). Individual and joint models and effect measure modification by preconception body mass index (BMI) were considered. Among 2,370 couples, 19.5% of females and 29.9% of males were 25(OH)D deficient. Females with sufficient status had a 28%-higher likelihood of livebirth than deficient females (95%CI 1.05-1.56). Female and male 25(OH)D status were associated with live birth among those with normal BMI (sufficient vs. deficient: female aRR 1.39, 95%CI 1.00-1.99; male aRR 1.51, 95%CI 1.01-2.25) and among obese female partners (sufficient vs. deficient: aRR 1.33, 95%CI 0.95-1.85). Couples whom both partners had higher 25(OH)D had increased likelihood of live birth (both not deficient vs. both deficient aRR 1.26, 95%CI 1.00-1.58). No associations were observed with pregnancy loss or semen quality. Similar results were found for all biomarkers except calcium. Preconception vitamin D and bioavailability impact fertility among couples seeking infertility therapy, likely unrelated to semen quality. BMI stratified analyses demonstrated heterogenous associations.