Actinomycete strains isolated from marine sediment samples collected at the Bay of Bengal coast of Puddcherry, India was screened for antiproliferative activity on three cancer cell lines by trypan blue staining and MTT assay. The potential isolate having antiproliferative activity was cultured on ISP - Veg media supplemented with sea water, pH 7.2, and the inoculated broth was maintained at 28° C for 7 days. The media and cultural conditions for maximal growth have been optimized under shake- flask conditions by measuring the dry weight of the mycelium. The maximal growth was attained with the use of optimized production medium, pH of 7.2 and incubation temperature of 27°C. Based on molecular taxonomic and phylogenetic characterization, the strain was identified as Rhodococcus and designated as Rhodococcus sp. VITSNK6. The 16 S rDNA nucleotide sequence from the 16 S rRNA partial gene sequence of the isolate was deposited in the GenBank under the sequence of the accession number FJ 973467. The ethyl acetate extract prepared from the supernatant of the culture broth was used for testing the antiprolifertive activity against HeLa (human cervical carcinoma), MDA-MB 231 (human breast adenocarcinoma), HepG2 (hepatocellular carcinoma) cells, HEK (Human embryonic kidney) and Vero (green monkey kidney) cell lines. The susceptibility of cancer cell lines to the extract was in the order of Hep G2> MDA-MB 231>HeLa with the IC50 values 22 μg/ml, 35 μg/ml and 45 μg/ml respectively. The extract treated cancer cells showed apoptosis mediated cell death and the viability of cancer cells was inversely proportional to the concentration of the extract used. The normal cell lines were less susceptible to inhibition than cancer cells. The results of this study showed that the secondary metabolite produced by the isolate has marked antiproliferative activity against cancer cells.