Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-β signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment. Display omitted •A living biobank of CAFs from NSCLC patients recapitulates clinical CAF heterogeneity•Therapeutic profiling of the NSCLC CAFs reveals three distinctive functional subtypes•Subtype I and II CAFs have high HGF and FGF7 expression and protect cancer cells•Subtype III CAFs associate with better clinical response and immune cell migration Hu et al. identify that cancer-associated fibroblasts (CAFs) derived from non-small cell lung cancer patients are functionally heterogeneous. These functional distinctions directly impact response to clinical anticancer treatment and associate with the tumor immune microenvironment. Thus, CAF functional heterogeneity defines a unique parameter for designing more personalized treatments.