Cancer-associated fibroblasts (CAFs), the predominant stromal cells in the tumor microenvironment (TME), play a critical role in the progression of solid tumors, including oral squamous cell carcinoma (OSCC). However, the molecular mechanisms by which OSCC-derived factors mediate CAF differentiation remain incompletely understood. This study investigates the role of the C-X-C motif chemokine ligand 1 (CXCL1), secreted by OSCC cells, in promoting CAF differentiation and its downstream impact on tumor progression. Gingival fibroblasts (GFs) were treated with conditioned medium (CM) from various OSCC cell lines to assess their potential to induce CAF differentiation. Proteomic analysis using liquid chromatography-mass spectrometry identified CXCL1 as a key factor highly secreted in SCC25-derived CM, which exhibited the strongest capacity to induce CAF differentiation. CXCL1 synergistically enhanced TGF-β1-induced differentiation of GFs into α-smooth muscle actin (αSMA)- and vimentin-expressing CAFs by approximately 1.5-fold, confirming its co-stimulatory function. Conversely, silencing its receptor CXCR2 reduced CAF marker expression by over 50%, indicating a strong inhibitory effect on CAF differentiation. In vivo, co-injection of SCC25 cells with GFs significantly promoted tumor growth and stromal CAF marker expression, whereas CXCR2 knockdown in GFs led to a ~ 40% reduction in tumor volume and reduced αSMA/vimentin-positive CAFs. These findings establish CXCL1 as a pivotal mediator of CAF differentiation through CXCR2-dependent signaling, and highlight that the CXCL1-CXCR2 axis is a promising therapeutic target for modulating stromal-tumor interactions in OSCC.