The Cancer Genome Atlas network has revealed that the 'mesenchymal' epithelial ovarian cancer (EOC) subtype represents the poorest outcome, indicating a crucial role of stromal cancer-associated fibroblasts (CAFs) in disease progression. The cooperative role of CAFs in EOC metastasis has long been recognized, but the mechanisms of stromal CAFs activation are still obscure. Therefore, we carried out an integrative analysis to identify the regulator genes that are responsible for CAFs activation in microdissected tumor stroma profiles. Here, we determined that myristoylated alanine-rich C-kinase substrate (MARCKS) was highly expressed in ovarian stroma, and was required for the differentiation and tumor promoting function of CAFs. Suppression of MARCKS resulted in the loss of CAF features, and diminished role of CAFs in supporting tumor cell growth in 3D organotypic cultures and in murine xenograft model. Mechanistically, we found that MARCKS maintained CAF activation through suppression of cellular senescence and activation of the AKT/Twist1 signaling. Moreover, high MARCKS expression was associated with poor patient survival in EOC. Collectively, our findings identify the potential of MARCKS inhibition as a novel stroma-oriented therapy in EOC.