cDNA sequence of the zeta-carotene desaturase gene involved in carotenoids synthesis was cloned from the yellow Petals of Helianthus annuus ‘MinKui 3’ using RT-PCR and RACE techniques, named as HaZDS (GenBank No. KF263657). The full length cDNA was 2069bp, and it included an open reading frame (ORF) of 1761bp corresponding to 587 amino acids. The deduced amino acid sequence analysis showed that HaZDS protein contained a chloroplastic transit peptide sequence in the N-terminal region, a typical amino oxidase domain and NAD(P) binding domain that included one characteristic sequence. The phylogenetic analysis demonstrated that HaZDS was more related to ZDS protein from Tagetes erecta and Chrysanthemum × morifolium, shared up to 96.4% and 90.1% homologues. Quantitative RT-PCR analysis indicated that HaZDS gene showed the highest transcript abundance in fully opened flower. HaZDS gene was highly expressed in ray florets, green tubular florets, bracts and leaves, but lowly expressed in black tubular florets. With the petals yellow deepening, the relative expression level of HaZDS gene gradually increased. It was suggested that HaZDS gene could involve in the regulation of flower color formation in transcription level of Helianthus annuus.