Excessive exposure of ultraviolet B (UVB) increases the production of reactive oxygen species (ROS) and causes damage in cells and skin tissue, resulting in various skin diseases. This study was designed to investigate the inhibitory effect of the chloroform fraction of Sargassum horneri methanol extract (SHMC) on oxidative stress induced by UVB irradiation in keratinocytes. As a result, SHMC increased cell viability in UVB-irradiated keratinocytes by reducing intracellular ROS production. In addition, SHMC suppressed apoptosis by inhibiting the apoptotic body formation and the sub-G1 DNA content via the regulation of expression levels of an anti-apoptotic protein Bcl-2 and pro-apoptotic proteins, Bax, p53, cleaved caspase-3 and cleaved PARP. Moreover, SHMC inactivated the NF-κB signaling activated by UVB irradiation. These results indicate that SHMC protects keratinocytes against oxidative stress by modulating via the inactivating of NF-κB signaling.