Ultraviolet A (UVA) irradiation is responsible for a variety of changes in cell biology. The purpose of this study was to investigate the effects of UVA irradiation on the stemness properties of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Furthermore, we examined the UVA-antagonizing effects of L-cysteine ethylester hydrochloride (ethylcysteine) and elucidated its action mechanisms. The results of this study showed that UVA reduced the proliferative potential and stemness of hAMSCs, as evidenced by reduced proliferative activity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and downregulation of OCT4, NANOG, and SOX2, stemness-related genes. The mRNA level of hypoxia-inducible factor (HIF)-1α, but not HIF-2α was reduced by UVA. Moreover, the knockdown of HIF-1α using small interfering RNA (siRNA) for HIF-1α was found to downregulate stemness genes, suggesting that UVA reduces the stemness through downregulation of HIF-1α. In addition, we examined the mechanisms underlying the UVA-mediated effects and found that UVA induced production of prostaglandin (PG) E2 and 3′-5′-cyclic adenosine monophosphate (cAMP), and that this effect was mediated through activation of activating protein-1 (AP-1) and nuclear factor-κB (NF-κB). The UVA effects were antagonized by ethylcysteine, and the effects were found to be mediated by reduced production of PGE2 through the inhibition of JNK and p42/44 MAPK. Taken together, these findings show for the first time that UVA regulates the stemness of hAMSCs and its effects are mediated by downregulation of HIF-1α via the activation of PGE2–cAMP signaling. In addition, ethylcysteine may be used as an antagonizing agent to mitigate the effects of UVA. J. Cell. Biochem. 113: 3681–3691, 2012. © 2012 Wiley Periodicals, Inc.