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The potassium inwardly rectifying channel, subfamily J, member 12 (<i>KCNJ12</i>) gene is a promising candidate for economic traits because of its crucial roles in myoblast development. Here, a missense mutation (Cys &gt; Arg) was first detected to be located in exon 3 of <i>KCNJ12</i> from three Chinese cattle breeds by DNA-pool sequencing. Then, we performed an association analysis of this single-nucleotide polymorphism (SNP) with stature in three Chinese cattle populations (<i>n</i> = 820). A significantly positive correlation was revealed by a reduced animal general linear model and the CC genotype was the most favorable in three breeds. Further, we measured the expression profile of the <i>KCNJ12</i> gene in various cattle tissues and primary bovine skeletal muscle cells. Ubiquitous expression with high abundance in muscle was observed. Further, in primary bovine skeletal muscle cells, the <i>KCNJ12</i> mRNA expression was gradually up-regulated in differentiation medium (DM) compared with that in growth medium (GM), suggesting that the <i>KCNJ12</i> gene is involved in bovine myocyte differentiation. Conclusively, the <i>KCNJ12</i> gene is a functional candidate gene which can be used as a molecular marker for cattle breeding.