Identifying strategies to increase cancer cell kill while sparing normal tissue is critically important in cancer chemotherapy. Choline kinase (Chk), the enzyme that converts choline to phosphocholine (PC), is elevated in cancer cells and presents a novel target for increasing cell kill. Here, we have examined the effects of transiently down-regulating Chk by small interfering RNA against Chk (siRNA-chk) on PC and total choline-containing compound (tCho) levels and on the viability/proliferation of estrogen receptor-negative and estrogen receptor-positive breast cancer cell lines and a nonmalignant mammary epithelial cell line. We investigated the effects of combination treatment with transient siRNA-chk transfection and the anticancer drug 5-fluorouracil (5-FU) in those cell lines. Microarray analysis of the invasive estrogen receptor-negative MDA-MB-231 cell line was done to characterize molecular changes associated with Chk down-regulation. Chk down-regulation decreased PC and tCho levels in the malignant cell lines, whereas the cell viability/proliferation assays detected a decrease in proliferation in these cells. In contrast, Chk down-regulation had an almost negligible effect on PC and tCho levels as well as cell viability/proliferation in the nonmalignant cell line. A combination of siRNA-chk with 5-FU treatment resulted in a larger reduction of cell viability/proliferation in the breast cancer cell lines; this reduction was evident to a much lesser degree in the nonmalignant cells. Microarray analysis showed that Chk down-regulation affected 33 proliferation-related genes and 9 DNA repair-related genes. Chk down-regulation with siRNA-chk may provide a novel alternative to enhance the effect of anticancer drugs in malignant cells.