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Abstract Background The accumulation of MWCNTs in the lung environment leads to inflammation and the development of disease similar to pulmonary fibrosis in rodents. Adverse Outcome Pathways (AOPs) are a framework for defining and organizing the key events that comprise the biological changes leading to undesirable events. A putative AOP has been developed describing MWCNT-induced pulmonary fibrosis; inflammation and the subsequent healing response induced by inflammatory mechanisms have been implicated in disease progression. The objective of the present study was to address a key data gap in this AOP: empirical data supporting the essentiality of pulmonary inflammation as a key event prior to fibrosis. Specifically, Interleukin-1 Receptor1 (IL-1R1) and Signal Transducer and Activator of Transcription 6 (STAT6) knock-out (KO) mice were employed to target inflammation and the subsequent healing response using MWCNTs as a model pro-fibrotic stressor to determine whether this altered the development of fibrosis. Results Wild type (WT) C57BL/6, IL-1R1 (KO) or STAT6 KO mice were exposed to a high dose of Mitsui-7 MWCNT by intratracheal administration. Inflammation was assessed 24 h and 28 days post MWCNT administration, and fibrotic lesion development was assessed 28 days post MWCNT administration. MWCNT-induced acute inflammation was suppressed in IL-1R1 KO mice at the 24 h time point relative to WT mice, but this suppression was not observed 28 days post exposure, and IL-1R1 KO did not alter fibrotic disease development. In contrast, STAT6 KO mice exhibited suppressed acute inflammation and attenuated fibrotic disease in response to MWCNT administration compared to STAT6 WT mice. Whole genome analysis of all post-exposure time points identified a subset of differentially expressed genes associated with fibrosis in both KO mice compared to WT mice. Conclusion The findings support the essentiality of STAT6-mediated signaling in the development of MWCNT-induced fibrotic disease. The IL-1R1 KO results also highlight the nature of the inflammatory response associated with MWCNT exposure, and indicate a system with multiple redundancies. These data add to the evidence supporting an existing AOP, and will be useful in designing screening strategies that could be used by regulatory agencies to distinguish between MWCNTs of varying toxicity.