Abstract Purpose: Mechanisms of primary resistance to inhibitors of the programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) signaling axis in non–small cell lung cancer (NSCLC) are still poorly understood. While some studies suggest the involvement of trophoblast cell surface antigen 2 (TROP2) in modulating tumor cell resistance to therapeutic drugs, its specific role in the context of PD-1/PD-L1 axis blockade is not definitively established. Experimental Design: We performed high-throughput analysis of transcriptomic data from 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy in two large randomized clinical trials. To confirm our results at the protein level, we complemented this transcriptional approach by performing a multiplex immunofluorescence analysis of tumor tissue samples as well as a proteomic profiling of plasma. Results: We observed a significant association of TROP2 overexpression with worse progression-free survival and overall survival on PD-L1 blockade, independent of other prognostic factors. Importantly, we found increased TROP2 expression to be predictive of survival in patients treated with atezolizumab but not chemotherapy. TROP2 overexpression was associated with decreased T-cell infiltration. We confirmed these results at the proteomic level both on tumor tissue and in plasma. Conclusions: Our results suggest an important contribution of TROP2 expression to the primary resistance to PD-L1 blockade in NSCLC. TROP2-biomarker–based strategy may be relevant in selecting patients with NSCLC who are more likely to benefit from a combination of immunotherapy and an anti-TROP2 agent.