Extrapolation of cell culture-based test results to in vivo effects is limited, as cell cultures fail to emulate organ complexity and multi-tissue crosstalk. Biology-inspired microphysiological systems provide preclinical insights into absorption, distribution, metabolism, excretion, and toxicity of substances in vitro by using human three-dimensional organotypic cultures. We co-cultured a human lung equivalent from the commercially available bronchial MucilAir culture and human liver spheroids from HepaRG cells to assess the potential toxicity of inhaled substances under conditions that permit organ crosstalk. We designed a new HUMIMIC Chip with optimized medium supply and oxygenation of the organ cultures and cultivated them on-chip for 14 days in separate culture compartments of a closed circulatory perfusion system, demonstrating the viability and homeostasis of the tissue cultures. A single-dose treatment of the hepatotoxic and carcinogenic aflatoxin B1 impaired functionality in bronchial MucilAir tissues in monoculture but showed a protective effect when the tissues were co-cultured with liver spheroids, indicating that crosstalk can be achieved in this new human lung–liver co-culture. The setup described here may be used to determine the effects of exposure to inhaled substances on a systemic level.