Flowers of safflower (Carthamus tinctorius L.) have been widely used in traditional Chinese medicine because of their ability to improve cerebral blood flow. The major active ingredients in safflower are flavonoids. To date, few flavonoid biosynthesis genes have been cloned from this species. In our previous research, four isoform chalcone isomerase genes were screened. In this study, a new chalcone isomerase gene (designated as CtCHI-N) was cloned from a safflower cDNA library. A 696-bp cDNA for CtCHI-N was cloned, and its sequence was subsequently analyzed. The recombinant protein was successfully expressed in a prokaryotic expression system, and the expression of CtCHI-N in different tissues and at different stages of flowering was investigated. The results showed that CtCHI-N was expressed in a tissue-specific manner, and the greatest expression level occurred during early flowering. In addition, flavonoids at different stages of flowering were also measured, and the expression of CtCHI-N was found to correlate with flavonoid biosynthesis during flowering. CtCHI-N was successfully cloned in the experiment, and expression analysis revealed that CtCHI-N might influence flavonoid biosynthesis during flowering in safflower.