Cancer-associated fibroblasts (CAFs) exhibit phenotypic heterogeneity with each functional state playing critical roles in tumor progression. Notably, subtypes like inflammatory CAFs (iCAFs), characterized by increased chemokine/cytokine secretion, and myofibroblast-like CAFs (myCAFs), characterized by enhanced extracellular matrix (ECM) deposition and increased actomyosin contractility, can undergo phenotypic switching in response to cues from the tumor microenvironment (TME) and therapeutic interventions. Elucidation of the signaling pathways associated with the diverse phenotypes could enable development of strategies to therapeutically reprogram CAFs. Through the analysis of single-cell RNA sequencing data from colorectal cancer (CRC) patients, we identified that the PI3K/mTOR and MAPK/ERK signaling pathways, among other pathways, are linked to the formation of myCAF and iCAF subtypes, respectively. Unbiased pharmacological interference of 12 distinct signaling pathways using three-dimensional (3D) human CRC-derived CAF cultures, ex vivo patient-derived tumor fragments, and mouse models further revealed the significance of PI3K/mTOR and MAPK/ERK signaling in CAF plasticity and functional behavior. PI3K/mTOR inhibition drove iCAF formation through compensatory FGF-2 release and FGFR1–JAK2–STAT3 activation, leading to chemokine/cytokine secretion that promoted tumor spheroid growth and neutrophil infiltration. Conversely, MEK inhibition induced a myCAF phenotype via interferon-dependent ROCK and JAK1 signaling, resulting in ECM production that enhanced tumor colony formation. In summary, these findings reveal a functional significance of PI3K/mTOR and MAPK/ERK signaling pathways in CAF plasticity and underscore how standard-of-care targeted therapies can directly influence CAF phenotypes in CRC.