Background and objectives Immune checkpoint inhibitors are increasingly used to treat various malignancies, but their application in patients with kidney transplants is complicated by high allograft rejection rates. Immune checkpoint inhibitor–associated rejection is a novel, poorly understood entity demonstrating overlapping histopathologic features with immune checkpoint inhibitor–associated acute interstitial nephritis, which poses a challenge for diagnosis and clinical management. We sought to improve the understanding of these entities through biopsy-based gene expression analysis. Design, setting, participants, & measurements NanoString was used to measure and compare the expression of 725 immune-related genes in 75 archival kidney biopsies, including a 25-sample discovery cohort comprising pure T cell–mediated rejection and immune checkpoint inhibitor–associated acute interstitial nephritis and an independent 50-sample validation cohort comprising immune checkpoint inhibitor–associated acute interstitial nephritis, immune checkpoint inhibitor–associated T cell–mediated rejection, immune checkpoint inhibitor–associated crescentic GN, drug-induced acute interstitial nephritis, BK virus nephropathy, and normal biopsies. Results Significant molecular overlap was observed between immune checkpoint inhibitor–associated acute interstitial nephritis and T cell–mediated rejection. Nevertheless, IFI27, an IFN-α–induced transcript, was identified and validated as a novel biomarker for differentiating immune checkpoint inhibitor–associated T cell–mediated rejection from immune checkpoint inhibitor–associated acute interstitial nephritis (validation cohort: P<0.001, area under the receiver operating characteristic curve =100%, accuracy =86%). Principal component analysis revealed heterogeneity in inflammatory gene expression patterns within sample groups; however, immune checkpoint inhibitor–associated T cell–mediated rejection and immune checkpoint inhibitor–associated acute interstitial nephritis both demonstrated relatively more molecular overlap with drug-induced acute interstitial nephritis than T cell–mediated rejection, suggesting potential dominance of hypersensitivity mechanisms in these entities. Conclusions These results indicate that, although there is significant molecular similarity between immune checkpoint inhibitor–associated rejection and acute interstitial nephritis, biopsy-based measurement of IFI27 gene expression represents a potential biomarker for differentiating these entities.