Purpose To assess the potential role of nuclear auto-antigenic sperm protein (NASP) in the cellular sensitivity to 5-Fluorouracil (5-FU) in breast cancer cells. Methods The expression of two NASP isotypes, namely somatic NASP (sNASP) and testis NASP (tNASP) in breast cancer lines were detected under 5-FU treatment using real-time polymerase chain reaction and western blot assays. NASP effect on cellular viability and apoptosis under 5-FU treatment were evaluated. The interaction between NASP and its downstream proteins were evaluated using the co-immunoprecipitation (Co-IP) assays. Results 5-FU significantly decreased the mRNA and protein expression levels of sNASP. Inhibition of sNASP increased cellular viability, colony formation ability, but reduced apoptosis in tested cell lines in response to 5-FU, which were reversed by sNASP over-expression. Further study reveals 5-FU disrupts sNASP/TNF receptor-associated factor 6 (TRAF6) complex, potentiates cellular sensitivity to 5-FU via NK-kB. Conclusion Our findings suggest sNASP is a novel molecular target having potential to overcome the resistance to 5-FU in breast cancer cells.