Hypericum perforatum is a valuable medicinal plant with anti-depressant activity. Hypericin is the major compound responsible for such activity. In the present study, the effect of four nano-capsulated hormones of 2,4-epibrassinolide, spermidine, salycilic acid and cycocel were investigated on the amount of hypericin based on HPLC analysis in two locations (Saman and Isfahan). For each hormone, the normal form was also compared with nano form. The expression patterns of the key genes (Hyp-1, pks1, pks2) for hypericin production was also evaluated using qRT-PCR. Moreover, GC-MS analysis was also performed for determination of the compounds in studied treatments. The major compounds were germacrene D (3.29–33.53%), β-caryophyllene (0-4.08) and α-longipinene (0-24.05%). In most cases, nano-hormones led to increase in these components. Significant changes were obtained in expression of key genes in hypericin synthesis as a result of nano-hormones treatments in Isfahan site. Overall, nano-hormones revealed higher increase in expression of all genes as compared with normal hormones in this site. The expression of Hyp-1, Pks1 and Pks2 was significantly increased using spermidine, 24-epibrassinolide and cycocel in Isfahan location in both nano-hormones and normal ones, while the expression of Hyp-1 was decreased in SA treatment in Isfahan location. Based on HPLC analysis, hypericin ranged from 0.21 in control to 0.51 mg 100 g− 1 DW in nano-SA in Isfahan site. Finally, the expression of the key genes were mostly elevated in colder climates and nano-form formulation.