The International Thermonuclear Experimental Reactor (ITER) is an international project aimed at the production of carbon-free energy through the use of thermonuclear fusion. During ITER operation, in case of a loss-of-vacuum-accident, tungsten nanoparticles (W-NPs) could potentially be released into the environment and induce occupational exposure via inhalation. W-NPs toxicity was evaluated on MucilAir™, a 3D in vitro cell model of the human airway epithelium. MucilAir™ was exposed for 24 h to metallic ITER-like milled W-NPs, tungstate (WO42−) and tungsten carbide cobalt particles alloy (WC-Co). Cytotoxicity and its reversibility were assessed using a kinetic mode up to 28 days after exposure. Epithelial tightness, metabolic activity and interleukin-8 release were also evaluated. Electron microscopy was performed to determine any morphological modification, while mass spectrometry allowed the quantification of W-NPs internalization and of W transfer through the MucilAir™. Our results underlined a decrease in barrier integrity, no effect on metabolic activity or cell viability and a transient increase in IL-8 secretion after exposure to ITER-like milled W-NPs. These effects were associated with W-transfer through the epithelium, but not with intracellular accumulation. We have shown that, under our experimental conditions, ITER-like milled W-NPs have a minor impact on the MucilAir™ in vitro model.