Central to NF-κB signaling pathways is IKKγ/NEMO, a regulatory subunit of the cytoplasmic IκB kinase (IKK) complex, which undergoes various posttranslational modifications, specifically phosphorylation, to regulate its function. Furthermore, Kaposi's sarcoma-associated herpesvirus (KSHV) FADD-like interleukin-1β (IL-1β) converting enzyme (FLICE) inhibitory protein (vFLIP) activates the NF-κB signaling pathway by directly interacting with IKKγ/NEMO. However, the exact functions of IKKγ/NEMO phosphorylation and its KvFLIP interaction in NF-κB activation remain elusive. Here, we report two novel phosphorylation sites of IKKγ/NEMO and their negative effect on the IKKγ/NEMO-mediated NF-κB signaling pathway. First, the Src family protein tyrosine kinases (SF-PTKs), including Src, Fyn, Lyn, and Fgr, interact with and phosphorylate tyrosine residue 374 (Y374) of IKKγ/NEMO. Mutation of the Y374 residue to phenylalanine (Y374F) specifically abolished SF-PTK-mediated tyrosine phosphorylation, leading to increased tumor necrosis factor alpha (TNF-α)-induced NF-κB activity. Moreover, our mass spectrometry analysis found that the serine 377 residue (S377) of IKKγ/NEMO underwent robust phosphorylation upon KvFLIP expression. Replacement of the IKKγ/NEMO S377 residue by alanine (S377A) or glutamic acid (S377E) resulted in a significant increase or decrease of NF-κB activity and TNF-α-mediated IL-6 cytokine production, respectively. Our study thus demonstrates that the Y374 or S377 residue located at the C-terminal proline-rich domain of human IKKγ/NEMO undergoes phosphorylation upon TNF-α treatment or KvFLIP expression, respectively, resulting in the suppression of IKKγ/NEMO activity to induce NF-κB activation. This study suggests the potential phosphorylation-mediated feedback negative regulation of IKKγ/NEMO activity in the NF-κB signaling pathway. IMPORTANCE Since unchecked regulation of NF-κB has been linked to uncontrolled proliferation and cell death, the downregulation of the NF-κB signaling pathway is as important as its activation. Specifically, the phosphorylation-mediated modification of IKKγ/NEMO is a critical regulatory mechanism of NF-κB activity. Here, we report two novel phosphorylations of IKKγ/NEMO and their negative effects on the NF-κB signaling pathway. First, the Src family protein tyrosine kinase interacts with and phosphorylates tyrosine residue 374 of IKKγ/NEMO, suppressing tumor necrosis factor alpha (TNF-α)-induced NF-κB activity. Additionally, Kaposi's sarcoma-associated herpesvirus (KSHV) FADD-like interleukin-1β (IL-1β) converting enzyme (FLICE) inhibitory protein (KvFLIP) expression induces a robust phosphorylation of the serine 377 residue of IKKγ/NEMO, resulting in a significant decrease of NF-κB activity. Our study thus demonstrates that the Y374 or S377 residue of IKKγ/NEMO undergoes phosphorylation upon TNF-α treatment or KvFLIP expression, respectively, resulting in the suppression of IKKγ/NEMO activity to induce NF-κB activation. This also suggests the potential phosphorylation-mediated feedback negative regulation of IKKγ/NEMO activity in the NF-κB signaling pathway.