Abstract: Objective To analyze the structure and function of human Cyclin D3 promoter. Methods Cyclin D3 promoter sequences were amplified by PCR method and the PCR products were inserted into pGL3-Basic vectors to construct the Cyclin D3 promoter-pGL3-Basic vectors (pD3-Δ). The pD3-Δ plasmids were transiently transfected into A2780 cells with the liposome. The promoter activities of pD3-Δ were detected by dual luciferase reporter assay to screen the key area of transcription and regulation. The regulation regions were assayed by TFSEARCH 1.3v to presume the key transcription factors. Results Cyclin D3 promoter sequences with different length were amplifi ed successfully by PCR and the cyclin D3 promoter-pGL3-Basic vectors（pD3-Δ）were well constructed. The dual luciferase reporter assay results implicated two positive (-1044bp to -766bp, -362bp to -197bp) and one negative (-766bp to -551bp) regulatory sequences located in Cyclin D3 promoter. TFSEARCH displayed that binding motifs of some transcription factors, such as C/EBP, SP1, AP1, HSF and MZF1, were contained in the regions. Conclusion Cyclin D3 promoter sequences have two positive (-1044bp to -766bp, -362bp to -197bp) and one negative (-766bp to -551bp) regulatory sequences which contain binding motifs of some transcription factors, such as C/EBP, SP1, AP1, HSF and MZF1, etc., and these transcription factors could regulate the promoter activity through binding motifs in A2780 cells.