Melatonin (N‐acetyl‐5‐methoxytryptamine) plays important roles in plant defences against a variety of biotic and abiotic stresses, including UV‐B stress. Molecular mechanisms underlying functions of melatonin in plant UV‐B responses are poorly understood. Here, we show that melatonin effect on molecular signalling pathways, physiological changes and UV‐B stress resistance in Arabidopsis. Both exogenous and endogenous melatonin affected expression of UV‐B signal transduction pathway genes. Experiments using UV‐B signalling component mutants cop1‐4 and hy5‐215 revealed that melatonin not only acts as an antioxidant to promote UV‐B stress resistance, but also regulates expression of several key components of UV‐B signalling pathway, including ubiquitin‐degrading enzyme (COP1), transcription factors (HY5, HYH) and RUP1/2. Our findings indicate that melatonin delays and subsequently enhances expression of COP1, HY5, HYH and RUP1/2, which act as central effectors in UV‐B signalling pathway, thus regulating their effects on antioxidant systems to protect the plant from UV‐B stress. Several studies have demonstrated that melatonin plays a role in UV‐B responses, however, the molecular mechanism whereby melatonin affects the UV‐B pathway was not clear. This study examined the function of melatonin in molecular signaling pathways, physiological changes, and UV‐B stress resistance under UV‐B radiation in Arabidopsis. Exogenous melatonin treatment experiment indicated that melatonin could enhance the transcriptional level of genes on UV‐B signaling pathway and ameliorate ROS damage caused by UV‐B stress. This result was verified in SNAT overexpressing lines and knock‐down mutant.