Using a previously created Mycobacterium avium genomic library with GFP-promoter trap in Mycobacterium smegmatis, we screened for genes that are upregulated upon infection of A. castellanii. Clones exhibiting a 2.5-fold or greater increase in GFP expression, out of a total of 10,000 clones, were selected for further examination. Upregulation was confirmed in subsequent experiments. A total of 20 clones showed an increase in expression 24 and 48 h after infection. Homologues were identified, and genes were found to encode for a variety of functions, including metabolic pathways, protein transcription and translation, and macromolecule degradation. Eight out of the 20 genes were found to be the same as those upregulated upon human macrophage infection. Five genes were selected to confirm upregulation in M. avium following amoeba infection, using real time PCR. All 5 genes were found to be upregulated at least 2.5-fold in M. avium. These results showed that the GFP promoter library in M. smegmatis is a valid system for studying gene upregulation in M. avium systems, and that many M. avium genes are commonly upregulated following macrophage and amoeba infection.