Drug interaction caused by inhibition of the cytochrome P450 especially CYP3A4, may result in clinically important adverse reactions due to a change in metabolism of the drugs. Nomilin, one of the limonoid compounds found abundantly in citrus species, was examined for its inhibition against recombinant human CYP3A4 activity. The enzyme activity was determined using the high-performance liquid chromatography (HPLC) technique to quantify the production of 6-hydroxytestosterone from testosterone. The results demonstrated that nomilin inhibited CYP3A4 activity with the IC50 value of 14.54 ± 2.77 μM. The inhibitory profile suggested the mixed type reversible inhibition with the apparent Ki value of 4.83 μM. Furthermore, nomilin was not a mechanism-based inhibitor of CYP3A4 activity. However, its apparent IC50 and Ki values were in the micromolar range, it was possible that nomilin and natural product containing nomilin could interfere CYP3A4-mediated metabolisms, and potentially lead to food-drug interaction problems. The in vivo study may be needed to assess this further.