In the Netherlands, there is no registry system regarding the livebirth prevalence of trisomy 21 (T21). In 2007, a national screening programme was introduced for all pregnant women, which may have changed the livebirth prevalence of T21. The aim of this study is to analyse trends in factors that influence livebirth prevalence of T21 and to estimate the livebirth prevalence of T21 for the period of 2000-2013. National data sets were used on the following: (1) livebirths according to maternal age and (2) prenatal testing and termination of pregnancy (ToP) following diagnosis of T21. These data are combined in a model that uses maternal age-specific risk on T21 and correction factors for natural foetal loss to assess livebirth prevalence of T21. The proportion of mothers aged ≥ 36 years has increased from 12.2% in 2000 to 16.6% in 2009, to gradually decrease afterwards to 15.2% in 2013. The number of invasive tests performed adjusted for total livebirths decreased (5.9% in 2000 vs. 3.2% in 2013) with 0.18% a year (95% CI: -0.21 to -0.15; p < 0.001). Following invasive testing, a higher proportion of foetuses was diagnosed with T21 (1.6% in 2000 vs. 4.8% in 2013) with a significant increase of 0.22% a year (95% CI: 0.18-0.26; p < 0.001). The proportion of ToP subsequent to T21 diagnosis was on average 85.7%, with no clear time trend. This resulted in a stable T21 livebirth prevalence of 13.6 per 10,000 livebirths (regression coefficient -0.025 (95% CI: -0.126 to 0.77; p = 0.60).