Onosma pachypoda Boiss. is a medicinal plant and belongs to Boraginaceae family. Enhancement of secondary metabolites using modern biotechnological methods such as tissue culture and hairy root induction by Agrobacterium rhizogenes can be important. In the present study, hairy root induction by A. rhizogenes strain A4 on the callus of O. pachypoda was examined via dry and immersion methods under different spectral LED lights. The effect of LED lights was also investigated on the concentration of flavonoid content in inoculated callus. The results showed that the highest percentage (50%) of hairy root induction was obtained by immersing the callus in the suspension of A. rhizogenes under dark conditions. The highest flavonoid content was observed in inoculated callus under blue LED light and dark conditions. Therefore, the immersion method is a suitable method for inoculation of callus to induce hairy roots in the plant. It seems that using of different spectral LED lights also affects flavonoid content in the callus of O. pachypoda.