Ultraviolet B (UVB) irradiation increases the risk of various skin disorders, resulting in apoptosis, autophagy and oxidative stress and thereby promoting the risk of skin photoaging and carcinogenesis. The use of photochemoprotectors including natural products with antioxidant properties represents an effective strategy for preventing UVB-induced skin injury. Isoorientin (Iso), as a flavonoid compound, could be extracted from several plant species and possesses multiple biological activities. However, its role in regulating UVB-induced skin damage is little to be reported. In the study, we found that Iso treatment could protect human dermal fibroblasts (HDFs) against the effects of UVB irradiation by improving cell viability, suppressing MMP1 and MMP3 expression, inhibiting oxidative stress and inducing autophagy. In addition, Iso reduced UVB-triggered apoptosis, as evidenced by the decreased Caspase-3 activity in vitro. Furthermore, Iso was functioned as reactive oxygen species (ROS) scavenger that markedly hindered c-Jun N-terminal kinases (JNK) signaling activation in UVB-treated HFDs. Importantly, promoting JNK activity restored matrix metalloproteinase (MMP)-1/3 expression in Iso-incubated cells with UVB stimulation. Meanwhile, UVB exposure to the skin of mice and subsequent topical application of Iso delayed the progression of skin damage, resulting in autophagy and blocking the JNK activation and ROS production. In conclusion, these results indicated the photoprotective role of Iso and demonstrated that Iso could also be potentially used as an agent against UVB-stimulated skin damage. Copyright © 2019. Published by Elsevier Inc.