The granulocyte colony-stimulating factor (GCSF) regulates the maturation, proliferation, and differentiation of precursor cells of neutrophilic granulocytes, and has been widely studied in several species. To investigate the function of variants of sheep GCSF (sGCSF), this study compared difference in their mRNA expression levels. Both the activity and mRNA expression level of GCSFv2 were higher than those of GCSFv1. Their sequences were aligned, which showed that they had the highest homology with bovine GCSF. Then, predicted ovine GCSF isoforms and their constant C-terminals were cloned and expressed, which were stably expressed in mammalian cells. After purification, all GCSF functions were different both in vitro and in vivo, and the GCSF C-terminal was best. These results indicated that the ability to stimulate both the proliferation and differentiation of progenitor cells and to activate the maturation of neutrophils could be used for research of efficacious non-antibiotic protein drugs. Furthermore, GCSF can be used as candidate target of genetic breeding to specifically improve sheep immunity.