Granulocyte colony-stimulating factor (G-CSF) is the major regulator of hemopoiesis and granulopoiesis. However, overexpression of G-CSF has been implicated in several important processes in tumor biology such as tumor growth, angiogenesis, and metastasis. Four different mRNA isoforms resulting from alternative splicing have been reported for G-CSF (transcript variants 1, 2, 3 and 4). The mRNAs and protein products of splice variants 1 and 2 have been isolated for the first time, from tumor cell lines. In the present study for the first time we isolated the G-CSF transcript variant 4 encoding G-CSF isoform D from a highly malignant tumor cell line (Mehr80) with overexpression of G-CSF. Both the full-length G-CSF isoform B and G-CSF isoform D were cloned from Mehr80 cell line, overexpressed in Escherichia coli as N-terminal glutathione-S-transferase fusion proteins in the form of inclusion bodies and affinity purified by the batch method using glutathione-Sepharose 4B resin. Both fusion proteins were successfully cloned and expressed. Folded recombinant proteins were solubilized from inclusion bodies using sarkosyl, Triton X-114 and CHAPS and purified. The purity of G-CSF isoforms was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and they were clearly detected in western blot analysis using anti-G-CSF polyclonal antibody. The G-CSF plays various roles in physiological and pathological conditions, however to date, the differential function of G-CSF isoforms remains unknown. Considering the fact that G-CSF isoform D was isolated from a highly malignant tumor cell line with overexpression of G-CSF, the role of this splice variant in tumorigenesis requires further investigation.