Cancer is one of the leading causes of death worldwide, accounting for about 10 million deaths a year, or nearly one in six deaths. The most common types of cancer are breast, colorectal, lung, and prostate cancers. Prolonged application of hormone drugs leads to the development of resistance. The development of agents with high activity against resistant cells is a challenge. It is important to create novel targeted compounds and search for active molecules among previously developed. The study aimed to evaluate the sensitivity of 4-hydroxytamoxifen-resistant cells to 5-fluorouracil (5-FU) and analyse the signalling pathways that are regulated by 5-FU in breast cancer cells. Antiproliferative activity was assessed by the MTT assay, immunoblotting was used to evaluate the expression of proteins in breast cancer cells. Activity of 5-FU was evaluated on parental MCF7 cells and a cell subline with resistance to 4-hydroxytamoxifen (HT), named MCF7/HT. The 4-hydroxytamoxifen-resistant cells showed high sensitivity to 5-FU. Expression of estrogen receptor α (a key driver of breast cancer growth) in MCF7 and MCF7/HT cells was not sensitive to 5-FU treatment. In both parental and resistant cells, 5-FU induces changes in the activity of several signalling proteins. 5-FU activated Akt, ERKs 1/2, and cyclin D1. The data suggest that 5-FU should be further investigated as a chemotherapeutic for hormone-resistant cancers; the combination of 5-FU with Akt and ERKs 1/2 inhibitors may be effective.