INTRODUCTION: Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis, limited treatment options, and high recurrence rates. Surgery and mitotane-based chemotherapy remain the standard of care, and new treatment strategies are needed. Iron oxide nanoparticles (IONPs) offer promise as theranostic agents due to their modifiability for selective uptake and imaging. METHODS: We investigated the uptake, toxicity, and impact on steroidogenesis of dopamine-coated Fe/Fe₃O₄ core-shell IONPs in three ACC cell lines (H295R, HAC-15, and MUC-1). Uptake was assessed using flow cytometry, confocal microscopy, and TEM. A multicellular transwell model including human endothelial cells (HUVEC) and primary monocytes was used to simulate physiological barriers to delivery. RESULTS: IONP uptake by ACC cells was concentration- and time-dependent, with optimal uptake at 10 µg/mL. Nanoparticles localised primarily to the cytoplasm and vesicular compartments. At this concentration, IONPs did not impair ACC cell viability, proliferation, metabolic activity, or forskolin/angiotensin II-stimulated steroidogenesis. Higher concentrations (≥20 µg/mL) led to aggregation and reduced viability in some cell lines. In the transwell model, primary monocytes and endothelial cells also avidly absorbed IONPs, reducing nanoparticle availability to ACC cells. CONCLUSION: ACC cells actively internalise IONPs without significant impairment of viability or steroidogenesis at pharmacologically relevant concentrations. However, non-specific uptake by monocytes and endothelial cells reduces delivery efficiency. These findings highlight the need for strategies to enhance tumour-specific targeting and improve biodistribution in future theranostic applications.