Sea hares have greatly attracted the interest of all those investigating chemical defense substances. Most of these substances are low molecular weight compounds derived from algal diets. in vitro anticancer effect of a 60 kDa protein isolated from the purple fluid of Aplysia dactylomela on four human cancer cell lines was investigated in this study. A 60 kDa protein was purified from secreted purple fluid of A. dactylomela, a sea hare from Persian Gulf. The protein purification procedure consisted basically of ammonium sulfate precipitation, ion exchange chromatography using DEAE–Sepharose and ultra-filtration method. in vitro antiproliferative and cytotoxic activity of the protein of interest were evaluated on L929, K562, HL60 and NB4 human cancer cell lines. The antiproliferative and cytotxic effects of 60 kDa protein on human cancer cell lines were measured by MTT assay. Results showed that the 60 kDa protein of the purple fluid of A. dactylomela exhibited the antiproliferative effect on human cancer cell lines, especially on NB4 cell line. It was maximally active at 0.5 – 1.5 μg/ml on NB4 cell line. Interestingly, the protein did not show significant cytotoxic effects.