SMAD3, the messenger of the transforming growth factor beta (TGF-β) signaling pathway, plays essential roles in myogenesis and osteogenesis and may relate to the regulation of body weight. In this study, a 17 bp indel (NC_007308: g.101893_101909insGAGGATGAGTGCTCCAG) in intron3 of the SMAD3 gene was detected in four Chinese cattle breeds (Qinchuan, Jiaxian, Nanyang and Caoyuan) by using DNA pool sequencing, and its effects on gene expression and growth traits were analyzed in Qinchuan and Caoyuan cattle. The results showed that the indel locus was significantly associated with SMAD3 transcriptional levels where II genotypes had a higher value than DD genotypes in Qinchuan (QC) cattle muscle tissue (P < 0.05). In addition, the locus was strongly associated with chest girth, chest width, rump length, hucklebone width and body weight in 2-year-old QC cattle (P < 0.05) and body weight (12 months), body height (18 months) and chest girth (18 months) in Caoyuan cattle (P < 0.5). To the best of our knowledge, this is the first evidence of the association between SMAD3 indel and cattle phenotype, and it may contribute to understanding the function of the indel, which could be a promising marker for beef cattle breeding.