Aim: Nanoparticle-based drug carriers hold great promise for the development of targeted therapies in pregnancy with reduced off-target effects. Here, we performed a mechanistic in vitro study on placental localization and penetration of gold nanoparticles (AuNPs) in dependence of particle size and surface modification. Materials & methods: AuNP uptake and penetration in human placental coculture microtissues was assessed by inductively coupled plasma-mass spectrometry, transmission electron microscopy and laser ablation-inductively coupled plasma-mass spectrometry. Results: Higher uptake and deeper penetration was observed for smaller (3–4 nm) or sodium carboxylate-modified AuNPs than for larger (13–14 nm) or PEGylate AuNPs, which barely passed the trophoblast barrier layer. Conclusion: It is possible to steer placental uptake and penetration of AuNPs by tailoring their properties, which is a prerequisite for the development of targeted therapies in pregnancy.