Active participation in care by parents and zero separation between parents and their newborns is highly recommended during infant hospitalization in the neonatal intensive care unit (NICU). To study the association of a family integrated care (FICare) model with maternal mental health at hospital discharge of their preterm newborn compared with standard neonatal care (SNC). This prospective, multicenter cohort study included mothers with infants born preterm treated in level-2 neonatal units in the Netherlands (1 unit with single family rooms [the FICare model] and 2 control sites with standard care in open bay units) between May 2017 and January 2020 as part of the AMICA study (fAMily Integrated CAre in the neonatal ward). Participants included mothers of preterm newborns admitted to participating units. Data analysis was performed from January to April 2021. FICare model in single family rooms with complete couplet-care for the mother-newborn dyad during maternity and/or neonatal care. Maternal mental health, measured using the Parental Stress Scale: NICU (PSS-NICU). Secondary outcomes included survey scores on the Hospital Anxiety and Depression Scale, Postpartum Bonding Questionnaire, Perceived Maternal Parenting Self-efficacy Scale, and satisfaction with care (using EMPATHIC-N). Parent participation (using the CO-PARTNER tool) was assessed as a potential mediator of the association of the FICare model on outcomes with mediation analyses. A total of 296 mothers were included; 124 of 141 mothers (87.9%) in the FICare model and 115 of 155 (74.2%) mothers in SNC responded to questionnaires (mean [SD] age: FICare, 33.3 [4.0] years; SNC, 33.3 [4.1] years). Mothers in the FICare model had lower total PSS-NICU stress scores at discharge (adjusted mean difference, -12.24; 95% CI, -18.44 to -6.04) than mothers in SNC, and specifically had lower scores for mother-newborn separation (adjusted mean difference, -1.273; 95% CI, -1.835 to -0.712). Mothers in the FICare model were present more (>8 hours per day: 105 of 125 [84.0%] mothers vs 42 of 115 [36.5%]; adjusted odds ratio, 19.35; 95% CI, 8.13 to 46.08) and participated more in neonatal care (mean [SD] score: 46.7 [6.9] vs 40.8 [6.7]; adjusted mean difference, 5.618; 95% CI, 3.705 to 7.532). Active parent participation was a significant mediator of the association between the FICare model and less maternal depression and anxiety (adjusted indirect effect, -0.133; 95% CI, -0.226 to -0.055), higher maternal self-efficacy (adjusted indirect effect, 1.855; 95% CI, 0.693 to 3.348), and better mother-newborn bonding (adjusted indirect effect, -0.169; 95% CI, -0.292 to -0.068). The FICare model in our study was associated with less maternal stress at discharge; mothers were more present and participated more in the care for their newborn than in SNC, which was associated with improved maternal mental health outcomes. Future intervention strategies should aim at reducing mother-newborn separation and intensifying active parent participation in neonatal care. Netherlands Trial Register identifier NL6175.