Core binding factor (CBF) is a family of heterodimeric transcription factors composed of a DNA-binding CBFα subunit and a non-DNA-binding CBFβ subunit, which plays critical roles in regulating hematopoiesis, osteogenesis and neurogenesis. In the present study, two genes encoding Runt (designed as CfRunt) and CBFβ (designed as CfCBFβ) were cloned and characterized from scallop Chlamys farreri. The full-length cDNA of CfRunt and CfCBFβ consists of 2128 bp and 1729 bp encoding a predicted polypeptide of 530 and 183 amino acids with a conserved Runt domain and CBFβ domain, respectively. Electrophoretic mobility shift assay demonstrated that the recombinant CfRunt protein (rCfRunt) exhibited solid ability to bind specific DNA, whereas rCfCBFβ could remarkably increase the DNA-binding affinity of rCfRunt. The mRNA transcripts of CfRunt and CfCBFβ could be detected in all tested tissues, especially in hemocytes, heart, hepatopancreas or muscle. After bacterial challenge, the circulating total hemocyte count (THC) of scallop reduced to the lowest level at 6 h (P < 0.05), and then it recovered gradually to the control level at 48–96 h, while the mRNA expressions of CfRunt and CfCBFβ were significant up-regulated between 6 and 48 h (P < 0.05). After CfRunt gene was silenced by RNA interference, the hemocyte renewal rate and circulating THC both decreased significantly (P < 0.05). However, following the RNA interference of CfRunt, the mRNA expression of CfRunt was significantly induced (P < 0.05) and the attenuated hemocyte renewal rate and circulating THC could be repaired partially by LPS stimulation in the CfRunt-silenced scallops. The results collectively indicated that CfRunt and CfCBFβ, as conserved transcription factors, played essential roles in regulating hemocyte production of scallop.