Multiple lines of evidence suggest that natural compounds can prevent skin ageing induced by ultraviolet light. Luteolin, a bioactive compound found in chilli, onion, broccoli, celery and carrot, has been reported to exhibit anti-photoageing effects in vitro. However, the molecular targets and mechanisms of luteolin are still poorly understood. In this study, we sought to investigate the effects of luteolin on UVB-induced photoageing and the molecular mechanisms involved, using HaCaT human keratinocytes and SKH-1 hairless mice. Luteolin was found to inhibit UVB-induced MMP-1 expression in HaCaT cells, as well as UVB-induced activation of AP-1, a well-known transcription factor targeting the MMP-1 promoter region, as well as c-Fos and c-Jun, which comprise the AP-1 complex. In contrast, Western blot data showed that UVB-induced phosphorylation of JNK, ERK and p90RSK was not inhibited by luteolin. In vitro kinase assay data revealed that luteolin significantly suppressed JNK1 and p90RSK activity, but not that of JNK2 and ERK2. Pull-down assays showed that luteolin binds JNK1 in an ATP-competitive manner and p90RSK2 in an ATP-independent manner. Luteolin also inhibited UVB-induced wrinkle formation and MMP-13 expression, a rodent interstitial collagenase in mouse skin, in vivo. Taken together, our observations suggest that luteolin exhibits anti-photoageing effects in vitro and in vivo and may have potential as a treatment for the prevention of skin ageing.