Breast cancer is the second most common cancer in the United States and consists of 30% of all new female cancer each year. PKC iota (PKC-ι) is a bonafide human oncogene and is overexpressed in many types of cancer, including breast cancer. This study explores the role of PKC-ι in regulating the transcription factor Jun proto-oncogene (c-Jun), pro-inflammatory cytokine Tumor Necrosis Factor-alpha (TNF-α), and the Mitogen-Activated Protein Kinase/Jun N-terminal kinase (MAPK/JNK) pathway, which also exhibits an oncogenic role in breast cancer. ICA-1S, a PKC-ι specific inhibitor, was used to inhibit PKC-ι to observe the subsequent effect on the levels of c-Jun, TNF-α, and the MAPK/JNK signaling pathway. To obtain the results, cell proliferation assay, Western blotting, co-immunoprecipitation, small interfering RNA (siRNA), immunofluorescence, flow cytometry, cycloheximide (CHX) chase assay, and reverse transcription quantitative PCR (RT-qPCR) techniques were implemented. ICA-1S significantly inhibited cell proliferation and induced apoptosis in both breast cancer cell lines. Treatment with ICA-1S and siRNA also reduced the expression levels of the MAPK/JNK pathway protein, c-Jun, and TNF-α in both cell lines. PKC-ι was also found to be strongly associated with c-Jun, via which it regulated the MAPK/JNK pathway. Additionally, ICA-1S was found to promote the degradation of c-Jun and decrease the mRNA levels of c-Jun. We concluded that PKC-ι plays a crucial role in regulating breast cancer, and the inhibition of PKC-ι by ICA-1S reduces breast cancer cell proliferation and induces apoptosis. Therefore, targeting PKC-ι as a potential therapeutic target in breast cancer could be a significant approach in breast cancer research.