The human genome includes four cytochrome P450 2C subfamily enzymes, and CYP2C8 has generated research interest because it is subject to drug–drug interactions and various polymorphic outcomes. To address the structure-functional complexity of CYP2C8, its catalytic activity was studied using a directed evolution analysis. Consecutive rounds of random mutagenesis and screening using 6-methoxy-luciferin produced two mutants, which displayed highly increased luciferase activity. Wild-type and selected mutants were expressed on a large scale and purified. The expression levels of the D349Y and D349Y/V237A mutants were ~310 and 460 nmol per liter of culture, respectively. The steady-state kinetic analysis of paclitaxel 6α-hydroxylation showed that the mutants exhibited a 5–7-fold increase in kcat values and a 3–5-fold increase in catalytic efficiencies (kcat/KM). In arachidonic acid epoxidation, two mutants exhibited a 30–150-fold increase in kcat values and a 40–110-fold increase in catalytic efficiencies. The binding titration analyses of paclitaxel and arachidonic acid showed that the V237A mutation had a lower Kd value, indicating a tighter substrate-binding affinity. The structural analysis of CYP2C8 indicated that the D349Y mutation was close enough to the putative binding domain of the redox partner; the increase in catalytic activity could be partially attributed to the enhancement of the P450 coupling efficiency or electron transfer.