Objective: The objective was to investigate how ultrasound screening for Down syndrome (DS) in the first trimester, compared with a routine ultrasound examination in the second trimester, affected Maternal–Fetal Attachment (MFA) in mid-pregnancy. Method: This study of 2026 pregnant women was a sub-study of a larger RCT aiming at evaluating the effect of fetal screening for Down syndrome (DS) by means of an ultrasound scan, including measuring fetal nuchal translucency in gestational weeks 12–14. Women were randomly allocated either to the intervention or to a control group where routine care with an ultrasound scan in gestational week 17–20 was offered. Data were collected by questionnaires before randomization and in gestational week 24. MFA was measured by a modified version of the Cranley Maternal–Fetal Attachment Scale (CMFAS). Results: The mean score of MFA was 3.50 in the intervention group and 3.44 in the control group (p = 0.04). The mean scores on all subscales were slightly higher in the intervention group, but only statistically significant regarding “Differentiation of self from fetus” (p = 0.01). Conclusion: Ultrasound screening for DS in the first trimester may have a modest positive effect on MFA in mid-pregnancy, compared with a ultrasound scan in the second trimester.