Abstract: In order to reveal the structure and function of buffalo ATF3 gene, the full sequence of the coding region in buffalo ATF3 gene was cloned, and the physicochemical properties, structure and function of the encoded product were analyzed by bioinformatics methods. In addition, the tissue differential expression of this gene was further detected by RT-qPCR. The results showed that the complete CDS length of buffalo ATF3 gene was 546 bp, encoding 181 amino acids. The amino acid sequence of buffalo ATF3 has more than 98.3% homology with that of common cattle, zebus, yak, goats and sheep. In the phylogenetic trees constructed based on both nucleotide and amino acid sequences, the buffalo clusters with the common cattle, zebus, yak, and other species of the bovine family. Buffalo ATF3 is a hydrophilic protein with no signal peptide and transmembrane region, but contains a conserved domain of bZIP, which is localized in the nucleus. The proportion of α-helix, β-turn, extended strand and random coil in the secondary structure of ATF3 protein was 51.93%, 8.84%, 6.08% and 33.15%, respectively. The tertiary structure was similar to that of human ATF3 protein（5vpe.2.A）, which reached 45%. ATF3 protein has transcriptional regulator activity and regulates genome transcription by selectively and non-covalently binding to specific double-stranded genomic DNA sequences within the cis-regulatory region. The relative expression level of buffalo ATF3 gene was high in muscle, mammary gland, heart and kidney, but low or even not expressed in the remaining tissues tested. And the expression of this gene in the mammary gland of lactating buffalo was significantly higher than that in non-lactating buffalo. The results indicated that the ATF3 gene might be associated with lactation traits in buffalo.