Objective To investigate the changes of gene expression in the cardiac outflow tract (OFT) in Cx43 knockout (Cx43-/-) mouse embryos, and to elucidate the genes involved in the myocardialization of proximal cardiae outflow tract septum. Methods The cDNA was retrotranscripted from RNA, which extracted from OFT tissues of both Cx43-/- and Cx43 wild type (Cx43+/+) mouse embryos on embryonic day (ED) 14.5. The biotin-labeled cRNA derived from the transcription of cDNA was fragmented as probes. The probes were hybridized with Affymetrix Mouse Genome 430 2.0 Array. Gene Array Scanner was used to screen the signals of hybridization, and the expression of genes was detected. Results Among the differentially expressed genes, there were 143 upregulated and 235 downregulated in Cx43 knockout OFT tissue compared with those in Cx43+/+ heart. Functions of proteins encoded by the altered genes encompassed all functional categories, with the largest percentage in genes involved in signaling pathways such as regulation of transcription, cell cycle, etc. Among the differentially expressed genes in the Cx43-/- heart, some were related with TGFβ/BMP signaling pathway, and Ssr1, Ptk2 and Bmp6 were related with conotruncal defects. These genes were verified by Real-time PCR, and the result was consistent with that of microarray (P<0.05).Conclusions Scaned by Gene Array and verified by Real-time PCR, genes related with TGFβ/BMP signaling pathway, and Ssr1, Ptk2 and Bmp6 were differentially expressed in ED 14.5 Cx43-/- OFT tissue, which may be involved in the myocardialization of proximal cardiac outflow tract septum.