In mammals, both runt-related transcription factor 3 (RUNX3) and c-Myc are the downstream effectors of transforming growth factor-β1 (TGF-β1) signaling to mediate various cellular responses. However, information of their interaction especially in fish is lacking. In the present study, grass carp (Ctenopharyngodon idella) runx3 (gcrunx3) cDNA was cloned and identified. Interestingly, opposing effects of recombinant grass carp TGF-β1 (rgcTGF-β1) on c-myc and runx3 mRNA expression were observed in grass carp periphery blood lymphocytes (PBLs). Parallelly, Runx3 protein levels were enhanced by rgcTGF-β1 in the cells. These findings prompted us to examine whether Runx3 can mediate the inhibition of TGF-β1 on c-myc expression in fish cells. In line with this, overexpression of grass carp Runx3 and Runx3 DN (a dominant-negative form of Runx3) in grass carp kidney cell line (CIK) cells decreased and increased c-myc transcript levels, respectively. Particularly, the regulation of Runx3 and Runx3 DN on c-myc mRNA expression was direct since they were presented in the nucleus without any stimulation. In addition, rgcTGF-β1 alone suppressed c-myc mRNA expression in CIK cells as in PBLs. Moreover, this inhibitory effect was also observed when grass carp Runx3 and Runx3 DN were overexpressed. These results strengthened the role of TGF-β1 signaling in controlling c-myc transcription. Taken together, TGF-β1-mediated c-myc expression was affected at least in part by Runx3, thereby firstly exploring the functional role of Runx3 in TGF-β1 down-regulation on c-myc mRNA expression in fish.