Severe insect defoliation induced non-lignified tracheids and thin-walled latewood tracheids in the defoliation year in Larix kaempferi. These anomalous tracheids had heterogenous lignin deposition of S2 layer. Severe insect defoliation induces the formation of light ring consisted of thin-walled latewood tracheids in larch trees, which would affect wood quality. However, the identity of the secondary wall layers affected by insect defoliation remains unclear. The aim of this study is to clarify the effects of insect defoliation on the formation of secondary cell walls of tracheids in Larix kaempferi with focus on the defoliation timing. We observed by light microscopy and transmission electron microscopy (TEM) the secondary cell walls of tracheids produced in a defoliation year in L. kaempferi trees on which needles were attacked in July (gypsy moth, GM samples) or August (larch sawfly, LS samples). Light microscopy observations revealed that GM samples produced non-lignified tracheids in the transition zone between earlywood and latewood, as well as thin-walled latewood tracheids, and non-lignified tracheids were observed near the cambial zone in LS samples following defoliation for two consecutive years. TEM observations revealed that the tracheids in both samples had anomalous layers in secondary walls such as thin S2 layers with heterogeneous lignin deposition. The production of non-lignified tracheids corresponded to defoliation date in both samples, unlike changes in the layer composition of anomalous tracheids. Our study suggests that changes in wood structure depend on the date of insect defoliation and that insect defoliation affects the formation of secondary cell walls of tracheids, presumably in response to inadequate photosynthate supply as a result of defoliation.