To explore efficient of different gene transfer methods and to express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in silk glands of transgenic silkworm, a novel transgenic vector containing a hGM-CSF controlled by silkworm fibroin heavy chain gene promoter (Pfib-H) with a neomycin resistance gene (neo) driven by BmNPV (B. mori nucleopolyhedrovirus) immediate-early gene ie-1 promoter (Pie-1) was constructed. The transgenic vector was transferred into the B. mori by using sperm-mediated gene transfer, particle delivery system gene transfer, eggs puncture gene transfer, and gonadal injection gene transfer, respectively, and the effectiveness of different transfer methods was investigated. The results showed that the ratio of fluorescence silkworm in G0 generation for four transform methods was 3.44%, 2.07%, 1.25% and 0.02%, respectively, indicating exogenous DNA can be introduced into silkworms eggs by the four methods mentioned above. The obtained transgenic silkworms by using sperm-mediated gene transfer were screened with neo and gfp genes as selecting markers, and verified with PCR and Southern blot. The hGM-CSF were expressed to about 13.67 ± 0.59 and 14.55 ± 0.65 ng per gram freeze-dried powder of silk glands from G5 and G8 generation transgenic silkworm, respectively. A specific 22 kD-band of hGM-CSF was detected in western blot. Furthermore, bioactivity assay indicated that the hGM-CSF expressed in posterior silk glands of transgenic silkworm could stimulate the proliferation of K562 cells. These results provide interesting information for further research of exogenous genes in the silk glands of transgenic silkworm by using diverse methods.