Phytosterols are a group of nonpharmacological alternatives to prevent or control dyslipidemias and cardiovascular disease. Increasing the phytosterol content in rapeseed oil is important to enhance daily phytosterol intake. However, the mechanisms of biosynthesis and regulation of phytosterol in rapeseed remain unclear. In this study, two representative rapeseed cultivars with extremely high (H286) and low (H174) phytosterol content were selected and treated with various concentrations (0.5−5.0 mM) of methyl jasmonate (MeJA). The results showed that treatment with 1 mM MeJA increased the phytosterol content of H174 and H286 by 17% and 27%, respectively. Based on the multiomics data, a gene‐phytosterol regulatory network was constructed. We deduced that MeJA down‐regulated the expression level of BnaA07.SCL15, BnaC05.MYB61, and BnaC03.AGL2, thereby promoting the phytosterol biosynthesis , and which were validated through the transient expression in tobacco. Notably, overexpression of Arabidopsis BnaA07.SCL15 exhibited a significant decrease in their phytosterol content. Additionally, an integrative analysis of the high‐resolution metabolome and transcriptome revealed that the accumulation patterns of 997 metabolites were highly correlated with their corresponding gene expression patterns. MeJA also significantly affected flavonoid biosynthesis, α‐linolenic acid metabolism, and amino acid metabolism. Furthermore, BnaA09.TT8 and BnaC09.TT8 were found to regulate of flavonoids. Overall, this study provides valuable insights into the phytosterol biosynthesis in rapeseed and offers a simple and effective approach for improving rapeseed quality.