The innate immune system is the first player involved in the recognition/interaction with nanomaterials. Still, it is not the only system involved. The co-evolution of the microbiota with the innate immune system built an interdependence regulating immune homeostasis that is poorly studied. Herein, the simultaneous interaction of iron-oxide nanoparticles (Fe-oxide NPs), immune cells, and the microbiota associated with the blood of the sea urchin Paracentrotus lividus was explored by using a microbiota/immune cell model in vitro-ex vivo and a battery of complementary tools, including Raman spectroscopy, 16S Next-Generation Sequencing, high-content imaging, NanoString nCounter. Our findings highlight the P. lividus immune cells and microbiota dynamics in response to Fe-oxide NPs, including i) morphological rearrangement and immune cell health status maintenance (intracellular trafficking increasing, no phenotypic alterations or caspase 3/7 activation), ii) transcriptomic reprogramming in immune cells ( Smad6, Lmo2 , Univin , suPaxB , Frizzled-7, Fgfr2 , Gp96 upregulation), iii) immune signaling unchanged (e.g., P-p38 MAPK, P-ERK, TLR4, IL-6 protein level unchanged), iv) enrichment in extracellular vesicle released in the co-culture medium, and v) a shift in the composition of microbial groups mainly in favor of Gram-positive bacteria (e.g., Firmicutes , Actinobacteria ),. Our findings suggest that Fe-oxide NPs induce a multi-level immune cell-microbiota response restoring homeostasis. • Fe-NP-exposed immune cells display active intracellular compartments and trafficking • NPs induce immune cells to upregulate transcripts controlling tolerogenic responses • Fe-oxide NPs stimulate an increase in extracellular vesicles in the co-culture medium • NPs promote a reprogramming in the microbiota in favor of the Gram-positive bacteria • A microbiota/immune cell conditioning is suggested upon Fe-NP exposure