Despite the success of therapies in lung cancer, more studies of new biomarkers for patient selection are urgently needed. The present study aims to analyze the role of galectin-3 (GAL-3) in the lung tumor microenvironment (TME) using tumorspheres as a model and explore its potential role as a predictive and prognostic biomarker in non-small cell lung cancer (NSCLC) patients. For in vitro studies, lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) primary cultures from early-stage patients and commercial cell lines were cultured, using tumorsphere-forming assays and adherent conditions for the control counterparts. We analyzed the pattern of secretion and expression of GAL-3 using reverse transcription-quantitative real-time PCR (RTqPCR), immunoblot, immunofluorescence, flow cytometry and immunoassay analysis. Our results using three-dimensional (3D) models of lung tumor cells revealed that soluble GAL-3 (sGAL-3) is highly expressed and secreted. To more accurately mimic the TME, a co-culture of tumorspheres and fibroblasts was used, revealing that GAL-3 could be important as an immunomodulatory molecule expressed and secreted in the TME, modulating immunosuppression through regulatory T cells (TREGS). In the translational phase, we confirmed that patients with high expression levels of GAL-3 had more TREGS, which suggests that tumors may be recruiting this population through GAL-3. Next, we evaluated levels of sGAL-3 before surgery in LUAD and LUSC patients, hypothesizing that sGAL-3 could be used as an independent prognostic biomarker for overall survival and relapse-free survival in early-stage LUAD patients. Additionally, levels of sGAL-3 at pretreatment and first response assessment from plasma to predict clinical outcomes in advanced LUAD and LUSC patients treated with first-line pembrolizumab were evaluated, further supporting that sGAL-3 has a high efficiency in predicting durable clinical response to pembrolizumab with an area under curve (AUC) of 0.801 (p=0.011). Moreover, high levels might predict decreased progression-free survival and overall survival to anti-PD-1 therapy, with sGAL-3 being a prognosis-independent biomarker for advanced LUAD.