The health benefits of tomato seed oil (TSO) have been suggested to be related to its antioxidant activity, although at the moment not much information is available on the antioxidant effects of TSO in biological systems. In this paper, we evaluated the antioxidant capacity of TSO using different spectrophotometrical antioxidant assays (LPSC, FRAP, αTEAC, DPPH). Moreover, we determined the ability of TSO in inhibiting oxidative stress in human cultured macrophages. The peroxyl radical scavenging LPSC assay was the most sensitive assay to detect the antioxidant capacity of the TSO, followed by the DPPH, FRAP, and αTEAC assay. TSO was able to counteract spontaneous and H(2)O(2)-induced oxidative stress in human macrophages, limiting intracellular ROS production and controlling oxidative stress signaling. In particular, TSO was able to decrease the phosphorylation of the MAPK ERK1/2, JNK, and p-38, activation of the redox-sensitive NF-kB, and expression of the heat shock proteins 70 and 90. When the antioxidant capacity of TSO was compared with that of purified lycopene, inhibition of ROS production by TSO was remarkably higher. This was due to the high content of other antioxidants in TSO, including (5Z)-, (9Z)-, (13Z)-, and (15Z)-lycopene isomers, β-carotene, lutein, γ-tocopherol, and α-tocopherol.