Abstract: Flavonoids, especially chalcones such as hydroxysafflor yellow A and carthamin are the main active ingredients of safflower. To study the biosynthesis pathway of safflower flavonoids is of great significance for the quality control of safflower. Chalcone synthase (CHS) is an enzyme that plays an important role in regulation of the synthesis of flavonoids. However, for the time being, the role of CHS is not yet clear in the biosynthesis of safflower flavonoids. As a plant signaling regulator, JA/MeJA can activate CHS gene expression in plants. CtCHS1, one of the CHS genes in safflower, was elucidated in our previous work. In our continuous search for CtCHSs functions from this plant, other CHS genes CtCHS2 and CtCHS4 in safflower were examined. The floret was stimulated with methyl jasmonate (MeJA) and the transcriptome expression of CtCHS2 and CtCHS4 was analyzed by qRT-PCR at different time points of 0, 3, 6, and 12 h after stimulation. Further metabolites under stimulation by MeJA were analyzed by UHPLC/Q-TOF-MS. The results showed that the expression of CtCHS4 in response to MeJA significantly increased at 3 and 6 h, while the expression of CtCHS2 showed a trend of decrease after induction. Meanwhile, the accumulation of rutin, hydroxysafflor yellow A, D-phenylalanine, kaempferol-3-O-β-rutinoside and carthamin increased obviously. Especially, accumulation of hydroxysafflor yellow A was increased significantly at 3, 6 and 12 h after induction (P ≥ 0.05 or 0.01), but the change in kaempferol, kaempferol-3-O-β-D-glucoside, luteolin, quercetin-3-β-D-glucoside was not significant. The accumulation of hydroxysafflor yellow A and carthamin was positively correlated with the expression abundance of CtCHS4 with Pearson correlation analysis method (r ≥ 0.8). The data suggest that CtCHS4 may be a key gene for forming hydroxysafflor yellow A and carthamin and plays an important role in the accumulation of safflower chalcones. The CtCHS4-pMAL-C5X recombinant vector was successfully expressed in BL21 (DE3) Plys to express the product naringenin in vitro under the catalytic substrates p-coumaryol-COA and malonyl-CoA. The results of this study provide a new insight into synthetic genes involved in flavonoids biosynthetic pathway to elucidate the biosynthesis pathway of safflower chalcones.