The nemamides are hybrid polyketide-nonribosomal peptides that are produced in two neurons of the nematode Caenorhabditis elegans and promote recovery from starvation-induced early larval stage arrest. Because the worm produces the nemamides in minute quantities and no total synthesis of these molecules has been reported, it has been difficult to study their mechanism of action. Here, by using a miniaturized assay, we demonstrate that nemamides purified from the worm exhibit antiproliferative activity against mammalian cancer cells. Moreover, we present the stereoselective synthesis of two nemamide analogs and evaluate their biological activity in the proliferation assay. Comparison of the NMR data of the synthetic analogs with those of the nemamides provides further confirmation of the absolute configuration of the nemamides, which was proposed previously based on NMR analysis, Marfey's method, model cyclic peptide synthesis, and CD spectroscopy. While the nemamides exhibit significant biological activity, the analogs have reduced potency, indicating that the lipid tail, polyene group, and methoxy group all play crucial roles in the antiproliferative properties of the nemamides. These findings offer valuable insights into the structure-activity relationships of the nemamides and lay the groundwork for future synthetic and mechanistic investigations of this unique class of natural products.