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<p><i>TP53</i> has been shown to be involved in lactation in cattle. However, the role of <i>TP53</i> in buffalo lactation remains unknown. To this end, we isolated and identified the complete coding sequence (CDS) of the <i>TP53</i> gene from the buffalo mammary gland and further analyzed its molecular characteristics, function, tissue differential expression, and single-nucleotide polymorphism (SNP). A transcript of this gene was cloned with a CDS length of 1161 bp, encoding a protein consisting of 386 amino acid residues. Bioinformatics analysis showed that buffalo <i>TP53</i> CDS and the physicochemical characteristics, conserved domains, structure, and function of its encoded protein are highly similar to those of other species in Bovidae. The buffalo TP53 protein contains an N-terminal activation domain, a DNA-binding domain, and a tetrameric domain, and it plays a functional role in the nucleus. <i>TP53</i> was found to express in all 11 detected buffalo tissues, and its expression in the heart, kidney, brain, muscle, and rumen during lactation was significantly higher than that during non-lactation (<span class="inline-formula"><i>p</i>&lt;0.05</span>), while in the liver, lung, and mammary gland, its expression was the opposite (<span class="inline-formula"><i>p</i>&lt;0.05</span>). Interference experiments in buffalo mammary epithelial cells (BuMECs) showed that <i>TP53</i> inhibits the expression of genes related to milk protein and milk fat synthesis through the PI3K–AKT–mTOR pathway. A synonymous nucleotide substitution (c.204C <span class="inline-formula">&gt;</span> T) was found in the <i>TP53</i> CDS of river buffalo, which is the CC homozygote in swamp buffalo. The results indicate that the <i>TP53</i> gene is involved in buffalo lactation by negatively regulating the synthesis of milk protein and milk fat.</p>