Abstract: Plants form a defense system that responds to environmental change during evolutionary adaptation. Flavonoids, as vital secondary metabolites in plants, are important indicators of plant chemical defense capability in the defense system. In this study, seedlings ofTaxus cuspidatafrom five provenances were transplanted to four sites (37°N—43°N). The flavonoid content in leaf epidermis of T. cuspidata seedlings was determined by Multiplex-3 (Force-A, France) non-destructively. We analyzed the change patterns of flavonoid content in the leaf epidermis of T. cuspidata seedlings which was influenced by climate factors in the test sites and provenances. The relative contribution rate of environmental change in the test sites to the changes of flavonol index (FLAV) and anthocyanin index (ANTH) of T. cuspidata seedlings were higher than that of environmental change in the provenances and the interaction effect between the test sites and the provenances. The explanatory rates of climate factors in the test sites on FLAV and ANTH (49.8%, 55.4%) were higher than those of climatic factors in the provenances (35.3%, 29.2%). The FLAV and ANTH of T. cuspidataseedlings decreased with the increase of latitude in the test sites. The results of variation partitioning of climate factors in the test sites indicated that illumination had the highest explanation rate for the changes of FLAV and ANTH. The results of variation partitioning of climatic factors in the provenances revealed that waterrelated factors had the highest explanation rate for the change of FLAV and ANTH. The environmental change in the test sites played a leading role in driving the change of flavonoid content in T. cuspidataseedlings.T. cuspidata seedlings had stronger chemical defense ability in low latitude test sites. Illumination played a dominant role in driving the changes of flavonoid content in T. cuspidata seedlings in the short term, whereas water played a dominant role in the long term.