Podophyllotoxin is an important secondary metabolite because of its remarkable anticancer and anti‐Condyloma acuminatum activities. Juniperus sabina L. (J. sabina) has been proven as a new plant source of podophyllotoxins. However, the mechanism of podophyllotoxins accumulation and synthesis in J. sabina is unclear, which greatly limits their applications. Based on this, transcriptomic and metabolomic analyses were performed on different developmental stages of J. sabina leaves to determine the molecular mechanism of the biosynthesis of podophyllotoxins in this study. Firstly, 8 podophyllotoxins and 54 differentially accumulated compounds were identified from metabolomic analysis of J. sabina leaves at different developmental stages. The total lignans and podophyllotoxin contents were determined by HPLC, clarifying that May to September was the critical period for the accumulation of podophyllotoxins in J. sabina leaves. Transcriptome and metabolic analysis showed that 47 DEGs encoded 16 key enzymes involved in podophyllotoxin biosynthesis, among which 8 key enzyme genes (PAL, HCT, CCR, C4H, DIR, PLR, OMT and CYP82) were identified. Furthermore, JsPLR was proven to be a key rate‐limiting enzyme cytoplasmic‐localized. JsPLR overexpression significantly increased matairesinol and podophyllotoxin content.. The findings of qRT‐PCR study indicated that the differential expression of JsPLR gene might be a key factor leading to the differential accumulation of podophyllotoxin. Overall, the study brings new insight into the accumulation and formation mechanisms of podophyllotoxins and the comprehensive development and utilization of J. sabina resources.