Astaxanthin has been shown to have antiproliferative activity on breast cancer and skin cancer cells. However, the high cost of production, isolation and purification of purified astaxanthin from natural sources or chemically synthetic methods limit its usage on cancer therapy. We show that astaxanthin could be produced by fermentating the Phaffia rhodozyma (Xanthophyllomyces dendrorhous) yeast cells with brewer malt waste using a 20 L B. Braun fermentor. The percentage composition of astaxanthin from the P. rhodozyma was >70% of total pigment as estimated by the high performance liquid chromatographic analysis. Furthermore, the antiproliferative activity of this P. rhodozyma cell extract (PRE) was demonstrated on breast cancer cell lines including the MCF-7 (estrogen receptor positive) and MDA-MB231 (estrogen receptor negative) by using the [3-(4,5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium] (MTS) assay. No apoptotic cell death, but growth inhibitory effect was induced after 48 h of PRE incubation as suggested by morphological investigation. Anchorage-dependent clonogenicity assay showed that PRE could reduce the colony formation potential of both breast cancer cell lines. Cell death was observed from both breast cancer cell lines after incubation with PRE for 6 days. Taken together, our results showed that by using an economic method of brewer malt waste fermentation, we obtained P. rhodozyma with a high yield of astaxanthin and the corresponding PRE could have short-term growth inhibition and long-term cell death activity on breast cancer cells.