The RelA (p65) is a subunit of nuclear transcription factor kappa B (NF-kappaB) and actively participates in expression of NF-kappaB-dependent genes involved in inflammation and tumorigenesis. Hence, the regulation of p65 is an important strategy to regulate those responses. In this study, we provide data that the dichlorophenyl derivative of 1,2,4-thiadiazolidine (known as P(3)-25) induced cell death in NF-kappaB-expressing and doxorubicin-resistant cells. P(3)-25 inhibited NF-kappaB DNA binding activity partially, but inhibited NF-kappaB-dependent genes expression completely. It inhibited phosphorylation of Rel A (p65) by inhibiting activity of protein kinase A (PKA). The PKA inhibition was independent of adenylate cyclase activity or cAMP level. The PKA activity decreased due to inhibition of catalytic subunit of PKA. P(3)-25 inhibited almost 80% PKA activity at 100 nM concentration, having an IC(50) at 10.5 nM. P(3)-25 potentiated different chemotherapeutic agents-mediated cell death. Our results suggest that P(3)-25 inhibits PKA activity followed by decreased phosphorylation of p65 and transcriptional activity of NF-kappaB thereby decreasing antiapoptotic proteins resulting in induction of apoptosis in NF-kappaB-expressing and doxorubicin-resistant cells. The study might help to understand the mechanism of P(3)-25-mediated apoptosis and to design it as new chemotherapeutic drug for tumor therapy.