PikC is a cytochrome P450 (CYP) monooxygenase that is involved in the biosynthesis of pikromycin. In this study, conditions for the overexpression of soluble PikC in Escherichia coli (E. coli) were optimized. More than 67.9% of the total PikC expressed was obtained in the soluble form when the cultures were induced at OD 600 of 3.84 with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and harvested after culturing at 20°C for 24 h. Under these conditions, 72, μg/mL of pikC could be obtained in the soluble fraction whereas 34, μg/mL was in the form of inclusion bodies. The factors controlling PikC expression and their interactions were studied according to the factorial design. Within the designed range, an increased IPTG concentration in the medium not only exerted a negative effect but also it interacted negatively with the induction time and induction period after the addition of TPTG. Furthermore, the latter two factors were found to interact negatively with one another.