Fumonisin B1 (FB1) is a toxic mycotoxin produced by Fusarium verticillioides, predominantly present in corn. The principal biochemical responses of FB1 involve disruption of sphingolipid metabolism from the inhibition of ceramide synthesis leading to accumulation of free sphingoid bases, particularly sphinganine. The ability of FB1 to modulate signal transduction pathways plays a role in its toxicity. We recently reported that FB1 selectively and transiently activates protein kinase Cα (PKCα) in porcine renal epithelial cells (LLC-PK1). The aim of current study was to investigate the effect of PKCα activation by FB1 on NF-κB activation and subsequently on TNFα gene expression and caspase 3 induction in LLC-PK1 cells. FB1 (1 μmol/L for 5 min) transiently activated PKCα and increased nuclear translocation of NF-κB, followed by their down-regulation at later time points. Preincubating the cells with the PKC inhibitor, calphostin C, prevented the activation of NF-κB by FB1. TNFα mRNA expression was increased after 15 min exposure to FB1 or the PKC activator, phorbol 12-myristate 13-acetate. In addition, an increase in caspase 3 activity was observed after addition of FB1 for 1 h. Calphostin C prevented both the FB1-induced increase in TNFα expression and caspase 3 activation. In summary, we hereby demonstrate that the FB1 activation of NF-κB and sequential induction of TNFα expression resulting in the subsequent increase in caspase 3 activity are all dependent on PKCα stimulation in LLC-PK1 cells.