Callus induction and proliferation of Echium italicum L. (Boraginaceae) were investigated using cotyledon, hypocotyl and root explants. Calli were initiated and established using B5, LS, 1/2LS and White media supplemented with different auxins, including 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA) and 1-naphthaleneacetic acid (NAA) in combination with kinetin. The maximum pigmented callus induction (100%) was observed in the White medium. The n-hexane extract of proliferated callus tissues were analysed by TLC and HPLC. The major secondary metabolite was separated by preparative HPLC and its structure was elucidated by UV, ¹H and ¹³C-NMR spectroscopy. As a result, shikonin acetate was identified by various spectroscopic methods from callus culture of E. italicum. These findings highlight the shikonin production potential of the E. italicum callus, which may be considered as a new source for the production of shikonin and its derivatives for industrial use.