As the function of autophagy becomes evident in a number of diseases, including cancer and infection, it is crucial to construct macrophage cell lines with stable expression of the microtubule-associated protein light chain 3 (GFP-LC3). In this study, a mouse LC3 open-reading frame was amplified by RT-PCR, and cloned into the pEGFP-C1 plasmid for expression of the GFP-LC3 fusion protein. The recombinant plasmid was transfected into RAW264.7 cells using Lipofectamine 2000 reagent and stably transfected clones were selected by G418 screening. Autophagic puncta formation was observed by fluorescense microscopy. Additionally, we found that starvation treatment induced a significant increase in the number of autophagosomes, while wortmannin treatment significantly repressed the formation of autophagosomes. This study indicated that the RAW264.7 cell line stably expressing GFP-LC3 is available for use in a GFP-LC3 puncta formation assay, and may contribute to basic investigations of autophagic function or drug screening targeted at autophagy.