BACKGROUND Selenium concentration of one spot urine sample cannot reflect selenium status during whole pregnancy. Studies on variations of urinary selenium concentration at different stages of pregnancy are limited. OBJECTIVE To assess variations of urinary selenium concentrations during three trimesters of pregnancy and to explore the influencing factors. METHODS This study included 2613 pregnant women from a birth cohort study (Wuhan, China) enrolled between October 2014 and October 2016. Selenium concentrations of urine samples collected at three trimesters were measured. We used a generalized linear mixed effects model to observe the changes in urinary selenium concentration during pregnancy and its influencing factors. RESULTS The median value (range) of gestational weeks at urine sample collection was 11 (9-14), 24 (15-27), and 34 (28-41) respectively. Urine selenium concentration varied across trimesters (geometric mean: 16.34, 17.65 and 18.83 μg/g creatinine, respectively), with an upward trend (β = -0.145, 95%CI: -0.164, -0.126) (β = -0.066, 95%CI: -0.083, -0.048). The concentrations of urinary selenium increased with the increasing of educational level [β (95%CI): ≤ 9 years = -0.105 (-0.163, -0.047); 10-12 years = -0.086(-0.126, -0.047); > 12 years = reference]. Pregnant women who rarely or only took multivitamins in the first trimester [β (95% CI): rarely = -0.076 (-0.144, -0.007); only in the first three months of pregnancy = -0.104 (-0.170, -0.038); always = reference], or were not exposed to passive smoking during pregnancy（β = -0.093, 95% CI: - 0.173, - 0.014), or exercised three to four days per week for the first three months before delivery（β = -0.074, 95% CI: - 0.140, - 0.008) had lower urinary selenium concentrations. Pregnant women who took calcium supplements after the third month of pregnancy had higher urinary selenium levels（β = 0.114, 95% CI: 0.059, 0.169). CONCLUSIONS Urine selenium concentrations of pregnant women showed an upward trend across trimesters. Educational level, calcium or multivitamin supplementation, passive smoking, and physical exercise might impact urine selenium levels.