Granulocyte colony-stimulating factor (G-CSF) affects granulopoiesis and is important for mobilizing neutrophils into blood circulation. Due to the hematopoietic properties of G-CSF, it has been widely used to clinically treat chemotherapy-induced neutropenia. However, G-CSF can promote tumors by inhibiting innate and adaptive immunity and enhancing angiogenesis and neoplastic growth. Most G-CSF-producing tumors are associated with a poor prognosis. This indicates that G-CSF promotes cancer progression. Thus, identifying regulatory molecules involved in tumor-derived G-CSF expression may provide therapeutic targets for cancer treatment. This study identified considerable G-CSF expression in malignant breast, lung and oral cancer cells. However, G-CSF expression was barely detectable in non-invasive cell lines. Expression of G-CSF mRNA and protein increased during exposure to tumor necrosis factor-α (TNF-α). Treatment with U0126 (a mitogen-activated protein kinase inhibitor) drastically reduced basal levels of G-CSF and TNF-α-induced G-CSF in aggressive cancer cells. This study also showed that knockdown of extracellular signal-regulated kinase (ERK) 2 by shRNA was necessary and sufficient to eliminate the expression of tumor-derived G-CSF. This did not apply to ERK1. Therefore, ERK2 (but not ERK1) is responsible for the transcriptional regulation of tumor-derived G-CSF. The results indicate the pharmaceutical value of specific ERK2 inhibitors in treating patients with G-CSF-producing tumors.