One of the leading causes of death worldwide is cancer. Excessive production of tumor necrosis factor (TNF)-α is known to activate nuclear transcription factor (NF)-κB, which plays a lethal role in the onset of multiple disorders including cancer. In this study, we aimed to determine the therapeutic role of novel gold nanoparticles conjugated with citrate (AuNPs-CIT) on the elevated expression of TNF-α in breast cancer cells. AuNPs-CIT were synthesized by the citrate-reduction method and were characterized by UV-VIS spectroscopic analysis, zeta-potential analysis, and size analysis. The potential of these newly generated AuNPs-CIT particles was tested on phorbol 12-myristate 13-acetate (PMA)-stimulated cancer cells. Our data showed that the AuNPs-CIT were spherical, with a mean size of 21.3±0.65 nm and a stabilized zetapotential at -41.4±0.98 mV. These newly generated AuNPs-CIT nanoparticles inhibited PMA-induced activation and nuclear translocation of NF-κB p65 in MCF-7 cells. They also have the tendency to block TNF-α expression in stimulated cancer cells. In conclusion, AuNPs-CIT inhibits PMA-induced TNF-α mRNA and protein expression via deactivation of NF-κB signaling in breast cancer cells. These findings suggest that AuNPs-CIT might be useful in cancer treatment.