A novel series of phenothiazine derivatives containing diethanolamine, methoxyethylamine, flavonoids, and a nitric oxide (NO) donor was designed and synthesized for the treatment of breast cancer. Phenothiazine derivatives (l) did not noticeably inhibit the growth of SUM159, MDA-MB-231, MCF-7, and SKBR-3 cells, whereas phenothiazine derivatives (ll) containing the NO donor were more potent or had comparable inhibitory activity to trifluoperazine (TFP) and thioridazine against SUM159, MDA-MB-231, MCF-7, and SKBR-3 cells. Compounds 20a-c and 21a-c showed the strongest activity in SUM159, MDA-MB-231, MCF-7, and SKBR-3 cells, and more potent inhibitory activity than TFP against KG1a cells (IC<sub>50</sub> = 1.63, 2.93, 1.14, 1.78, 2.20, and 1.20 vs. 4.58 μM). Compounds 20a and 21a had lower toxicity than compounds 20b-c and 21b-c, and inhibited colony formation in MCF-7 cells, decreased the formation of mammospheres in SUM159 cells, and inhibited the migration of MDA-MB-231 cells. Compounds 20a and 21a could inhibited pNF-κB-p65 as shown by dual-luciferase reporter assays and western blotting in MDA-MB-231 cells.