The National Research Council’s vision of using adverse outcome pathways (AOPs) as a framework to assist with toxicity testing to meet regulatory demands of new and emerging chemicals has continued to gain traction since its initial release. The need to grow the current AOP knowledge base has gained urgency, with the US EPA’s intent to eliminate animal toxicity testing by 2035. To meet these needs, an AOP for male rat renal tumors that occur through the ability of a chemical to induce α2u-N was organized based on extensive evidence of a specific mode of action (MoA) induced by multiple structurally diverse chemicals, including fuel additives (2,2,4-trimethylpentane), pesticides (1,4-dichlorobenzene), drugs (gabapentin), and solvents (decalin). This MoA is not relevant in humans, so it can be differentiated from other renal tumor MoAs using in vitro assays designed to measure the molecular initiating event (MIE) and/or key events (KEs) within an accepted AOP. Following identification and review of all relevant literature, including in vivo and in vitro studies, both the MIE and subsequent KEs were identified. The MIE is chemical binding to α2u, with KE1 being the inhibition of α2u catabolism due to chemical binding. This inhibition of α2u catabolism leads to its accumulation in the proximal tubule, resulting in the formation of protein droplets that crystalize (KE2). Exacerbated accumulation results in KE3 which is cytotoxicity, and compensatory cell proliferation. Atypical hyperplasia, along with lesions characteristic of protein accumulation, such as granular cast formation and linear mineralization, are considered to be KE4. With chronic exposure, a low incidence of renal adenoma and carcinoma development is seen in male rats. Based on the weight of evidence from the various evidence streams, the confidence in this AOP is high, and it could be used to assist with hazard identification in regulatory applications.