The role of phytochemicals in preventive and therapeutic medicine is a major area of scientific research. Several studies have illustrated the mechanistic roles of phytochemicals in Nrf2 transcriptional activation. The present study aims to examine the importance of the transcription factor Nrf2 by treating peritoneal macrophages from Nrf2(+/+) and Nrf2(-/-) mice ex vivo with phenethyl isothiocyanate (PEITC) and curcumin (CUR). The peritoneal macrophages were pretreated with the drugs and challenged with lipopolysaccharides (LPSs) alone and in combination with PEITC or CUR to assess their anti-inflammatory and antioxidative effects based on gene and protein expression in the treated cells. LPS treatment resulted in an increase in the expression of inflammatory markers such as cycloxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in both Nrf2(+/+) and Nrf2(-/-) macrophages, detected by quantitative polymerase chain reaction (qPCR). Nrf2(+/+) macrophages treated with PEITC and CUR exhibited a significant decrease in the expression of these anti-inflammatory genes along with an increase in the expression of hemeoxygenase-1 (HO-1), which is an antioxidative stress gene downstream of the Nrf2 transcription factor battery. Although there was no significant decrease in the expression of the anti-inflammatory genes or an increase in HO-1 expression in Nrf2(-/-) macrophages treated with either PEITC or CUR, there was a significant decrease in the protein expression of COX-2 and an increase in the expression of HO-1 in Nrf2(+/+) macrophages treated with PEITC compared to that with CUR treatment. No significant changes were observed in the macrophages from knockout animals. Additionally, there was a significant decrease in LPS-induced IL-6 and TNF-α production following PEITC treatment compared with that following CUR in Nrf2(+/+) macrophages, whereas no change was observed in the macrophages from knockout animals. The results from qPCR, western blot, and ELISA analyses in macrophages from Nrf2(+/+) and Nrf2 (-/-) mice indicate that Nrf2 plays an important role in the anti-inflammatory and antioxidative effects of PEITC and CUR, as observed by their decreased activities in Nrf2(-/-) macrophages.