The goal of this study was to elucidate differential expression of small RNAs along a natural CO2 gradient in the sea anemone Anemonia viridis and their possible involvement in transcriptional regulation in the response to ocean acidification conditions. Nine individual adult polyps were sampled off Vulcano Island, Italy, from a natural CO2 gradient of decreasing pH nearby a volcanic vent. Three different sampling locations were chosen for this study, representing seawater pH conditions 8.2, 7.9, and 7.6. In addition to small RNA sequencing, DNA sequencing was performed from one individual at pH 8.2 to be able to identify small RNA precursors.