Cell suspension cultures of Lithospermum erythrorhizon are capable of producing a large amount of shikonin derivatives (red naphthoquinone pigments specific to the roots) when transferred to pigment production medium M9, although the pigment production fails in Linsmaier and Skoog’s (LS) liquid medium used for subculture. Shikonin is biosynthesized by coupling of p-hydroxybenzoic acid (PHB) derived from shikimate pathway, and geranyldiphosphate from mevalonate pathway. We have been studying the regulatory mechanism of shikonin biosynthesis on chemical and biochemical level, as well as molecular genetic level. In this paper, regulation of PHB accumulation, one of the key intermediate of shikonin biosynthesis, and gene expression of two enzymes, phenylalanine ammonia-lyase and 4-couma-rate: CoA ligase, involved in the biosynthetic pathway of shikonin are elucidated. In recent years, it was also found that Lithospermum cell cultures synthesize benzoquinone derivatives, and phenylpropanoids as end products, adding shikonin. The biosynthetic regulation of the branching point between shikonin and benzofurans is also discussed.