Of various metal ions (Ca2+, Cr3+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+ and Zn2+) added to the culture medium of Agrobacterium tumefaciens at 1 mM, only Ca2+ increased Coenzyme Q10 (CoQ(10)) content in cells without the inhibition of cell growth. In a pH-stat fed-batch culture, supplementation with 40 mM of CaCO3 increased the specific CoQ(10) content and oxidative stress by 22.4 and 48%, respectively. Also, the effect of Ca2+ on the increase of CoQ(10) content was successfully verified in a pilot-scale (300 L) fermentor. In this study, the increased oxidative stress in A. tumefaciens culture by the supplementation of Ca2+ is hypothesized to stimulate the increase of specific CoQ(10) content in order to protect the membrane against lipid peroxidation. Our results improve the understanding of Ca2+ effect on CoQ(10) biosynthesis in A. tumefaciens and should contribute to better industrial production of CoQ(10) by biological processes.