Tumor necrosis factor alpha (TNF-alpha) plays a paramount role in homeostasis by inducing tumor cytotoxicity and activating immune system. The signaling complexes formed by TNFR1 to activate JNK, p38, and nuclear factor-kappa B pathways and to subsequently induce apoptosis and necroptosis are well known. However, this "canonical TNF-alpha signaling" does not explain how ERK, AKT, and STAT3 can be activated by TNF-alpha. In addition, little to nothing is known about negative regulation of TNFR1 signaling. Because cyclic AMP-activated kinase (PKA) shows anti-TNF and anti-inflammatory activities, we postulated that PKA might affect TNF-alpha signaling by directly phosphorylating TNFR1. In line with this, we identified 2 putative PKA-phosphorylation motifs RRRT411 and REAT(417) within the death domain of TNFR1, and investigated whether "canonical" and "noncanonical" TNFR1 signaling is regulated by modifications of T411 and T417. In this study, we demonstrate for the first time that PKA directly binds to and phosphorylates TNFR1 after TNF-alpha stimulation. To further support our hypothesis, we generated alanine and phosphomimetic (aspartic acid) mutants of TNFR1 at positions T411 and T417, ectopically expressed these mutants, and determined their influence on TNF-alpha-induced activations of ERKs, AKT, STAT3, p38 alpha, and JNK1/2. Our results clearly showed that phosphomimetic mutants significantly suppressed and alanine mutants augmented TNF-alpha-induced phosphorylations of ERKs, AKT, Stat3, p38 alpha, and JNKs. These findings strongly suggest that PKA-mediated phosphorylation of T411 and T417 of TNFR1 interferes with both "canonical" and "noncanonical" TNF-alpha signaling.