This study was performed to screen and select Lactobacillus strains from chicken feces for probiotic use in animals. Of these strains, strain A12 had the highest immunostimulatory effect. Therefore, strain A 12 was characterized as a potential probiotic. Strain A12 was tentatively identified as Lactobacillus acidophilus A12, using the API 50 CHL kit based on a 99.9% homology. L. acidophilus A12 was highly resistant to artificial gastric juice (pH 2.5) and bile acid (oxgall). Based on results from the API ZYM kit, leucine arylamidase, crystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, alpha-galactosidase, beta-galactosidase, beta-glucosidase, P-glucosidase, and N-acetyl-beta-glucosamidase were produced by strain A12. L. acidophilus A12 showed resistance to several antibiotics (nisin, gentamicin, and erythromycin). The amount of interleukin (IL)-1 alpha in 20x concentrated supernatant from L. acidophilus A12 was approximately 156 pg/ml. With regard to antioxidant activity, L. acidophilus A12 supernatant showed 60.6% DPPH radical scavenging activity. These results demonstrate the potential use of L. acidophilus A12 as health-promoting probiotics.