To investigate the protective effects of acteoside against apoptosis induced by X-ray radiation in human skin fibroblasts (HSFs), the cells were divided into the following groups: Control group; X-ray radiation group; acteoside group, in which the confluent cells were preincubated with 50 μg/ml acteoside for 2 h followed by radiation; and positive control group, in which the cells were preincubated with 50 μg/ml paeoniflorin followed by radiation. For the radiation, HSF cells preincubated with acteoside or paeoniflorin were exposed to X-ray beams at a dose-rate of 3 Gy/min (16 Gy in total). Cell viability, apoptosis and intracellular alteration of redox were monitored by MTT and flow cytometry. Compared with the radiation group, the number of cells arrested at the G0/G1 phase was significantly reduced in the acteoside and paeoniflorin groups, respectively (P<0.05). X-ray radiation induced marked apoptosis in HSF cells and acteoside reversed this effect. Compared with the radiation group, the generation of intracellular reactive oxygen species (ROS) was abrogated by pre-incubation with acteoside or paeoniflorin (P<0.05). In addition, the upregulation of pro-caspase-3 induced by radiation was reversed by acteoside or paeoniflorin. Radiation could induce upregulation of Bax and downregulation of Bcl-2; however, it was reversed completely after administration of acteoside or paeoniflorin. Furthermore, the enhanced expression of ERK and JNK induced by radiation was reversed by acteoside or paeoniflorin. Acteoside could protect the cells from X-ray induced damage through enhancing the scavenging activity of ROS, decreasing the Bax/Bcl-2 ratio and downregulating the activity of procaspase-3, as well as modulating the mitogen-activated protein kinase signaling pathways.