The objective of this study was to determine the fatty acid composition and the antiproliferative effect of extracts and fractions from Euphorbia supina. With regards the fatty acid composition, the percentages of 18:3n-3 in acetone/methylene chloride (A+M) and methanol (MeOH) extracts were 53.4 and 42.1%, respectively. Among the fractions, an 85% aqueous methanol (85% aq. MeOH) fraction contained the highest percentage of 18:3n-3. Treatments with crude extracts and fractions significantly inhibited the growth of HT-29 and AGS human cancer cell lines (p<0.05). The A+M extract showed a higher inhibitory effect on the growth of both cancer cells compared to MeOH extract. Among the fractions, the 85% aq. MeOH and n-hexane fractions exerted a greater inhibitory effect on the proliferation of both types of cancer cells. Our results suggest that 85% aq. MeOH and n-hexane fractions exert potent inhibitory effects on the proliferation of human cancer cells.