Objective To explore the antagonistic effect of nucleic acid aptamer on cytotoxicity of palytoxin (PLTX). Methods The cytotoxicity of PLTX on mouse embryonic fibroblast cell line NIH-3T3, Chinese hamster ovary cell line CHO-K1 and immortalized human keratinocyte cell line HaCaT and the antagonistic effect of aptamer 13-S3 to PLTX (with the concentration ratio of 1∶1) were detected and compared by cytotoxicity experiment. The hemolytic effect of PLTX on sheep erythrocytes and the inhibitory effect of aptamer on the hemolytic activity of PLTX were detected by erythrocyte hemolysis test. Results The cytotoxicity test showed that the half inhibitory concentration (IC₅₀) of HaCaT cells was (1.24±0.93) nmol/L, that of CHO-K1 cells was (3.95±0.78)×10^-3 nmol/L, and that of NIH-3T3 cells was (5.18±1.92)×10^-3 nmol/L. Compared with the PLTX control group, the survival rates of HaCaT cells were significantly increased in different concentration (9.3×10^-4, 9.3×10^-3, 9.3×10^-2, 9.3×10^-1, 9.3, and 93.3 nmol/L) PLTX＋aptamer 13-S3 groups (all P＜0.05). At the concentration of 12.5 nmol/L PLTX, compared with the PLTX＋aptamer N7-T (the specific aptamer of nodularin-R) group, the hemolysis rate of PLTX＋aptamer 13-S3 group decreased more significantly (P＜0.05), indicating that the aptamer 13-S3 could specifically inhibit the hemolytic activity of PLTX on sheep erythrocytes. Conclusion The survival rate of HaCaT cells has a good gradient response to the change of PLTX concentration (9.3×10^-4-93.3 nmol/L), and corresponding concentration of nucleic acid aptamers can antagonize the cytotoxicity of PLTX on HaCaT cells. At a concentration of 12.5 nmol/L PLTX, the corresponding concentration of aptamer can effectively inhibit the hemolytic activity of PLTX on sheep erythrocytes and play a cytoprotective role.