The epidermal growth factor (EGF) receptor (EGFR) overexpressed in many cancers, including lung and head and neck cancers, and is involved in cancer cell progression and survival. PD-L1, increases in tumor cells to evade and inhibit CD8+ T cells, is a clinical immunotherapeutic target. This study investigated the molecular mechanism of EGF on regulating PD-L1 in EGFR-positive cancers and determined potential agents to reduce PD-L1 expression. RNA sequencing (RNAseq) and bioinformatics analysis were performed to determine potential driver genes that regulate PD-L1 in tumor cells-derived tumorspheres which mimicking cancer stem cells. Then, the specific inhibitors targeting EGFR were applied to reduce the expression of PD-L1 in vitro and in vivo. We validated that EGF could induce PD-L1 expression in the selected EGFR-positive cancers. RNAseq results revealed that STAT1 increased as a driver gene in KOSC-3-derived tumorspheres; these data were analyzed using PANTHER followed by NetworkAnalyst. The blockade of EGFR by afatinib resulted in decreased STAT1 and IRF-1 levels, both are transcriptional factors of PD-L1, and disabled the IFNr-STAT1-mediated PD-L1 axis in vitro and in vivo. Moreover, STAT1 knockdown significantly reduced EGF-mediated PD-L1 expression, and ruxolitinib, a JAK1/JAK2 inhibitor, significantly inhibited STAT1 phosphorylation to reduce the IFNr-mediated PD-L1 axis. These results indicate that EGF exacerbates PD-L1 by increasing the protein levels of STAT1 to enforce the IFNr-JAK1/2-mediated signaling axis in selected EGFR-positive cancers. The inhibition of EGFR by afatinib significantly reduced PD-L1 and may be a potential strategy for enhancing immunotherapeutic efficacy.