Background T helper 17 (Th17) cells actively participate in the tumor immune response in lung cancer. However, the heterogeneity and plasticity of intratumoral Th17 cells in lung cancer remain elusive. In this study, Th17 subpopulations were characterized in a mouse lung cancer model. Methods Urethane was administered to induce lung cancer in interleukin (IL)-17-EGFP transgenic mice. Flow cytometry was used to analyze the phenotypes, signaling status, and functions of Th17 subpopulations either in vivo or in vitro. The adoptive transfer assay and real-time polymerase chain reaction were applied to analyze the plasticity of Th17 subpopulations. Results IL-6R alpha(high)CD27(-) Th17 and IL-6R alpha(low)CD27(+) Th17 were identified in intratumoral Th17 cells. The two subpopulations expressed equivalent ROR gamma t. However, the former expressed higher T-bet but lower Foxp3, more IL-17A and IFN-gamma but less IL-10 than the latter. Furthermore, IL-6R alpha(high)CD27(-) Th17 moderately inhibited the proliferation of lung cancer cells while IL-6R alpha(low)CD27(+) Th17 could not. IL-6R alpha(high)CD27(-) Th17 exhibited weaker Jun N-terminal kinases (JNK) signaling but stronger signal transducer and activator of transcription 3 (Stat3) signaling than IL-6R alpha(low)CD27(+) Th17. The adoptive transfer assay indicated that both subpopulations downregulated ROR gamma t in recipients' spleens but maintained ROR gamma t in recipients' lungs. Meanwhile, IL-6R alpha(high)CD27(-) Th17 expressed higher T-bet and IFN-gamma than IL-6R alpha(low)CD27(+) Th17 in the recipients. IL-6R alpha(low)CD27(+) Th17 expressed Foxp3 and IL-10 in recipients' spleens but not lungs. Conclusions This study reveals intratumoral Th17 subpopulations with distinct functional properties and signaling patterns, thus offering valuable insight into Th17 heterogeneity and plasticity in lung cancer.