Inflammatory bowel disease (IBD), encompassing Ulcerative Colitis (UC) and Crohn’s Disease (CD), is characterized by chronic gastrointestinal inflammation driven by genetic, environmental, immune, and microbial factors. Acetyl-CoA Synthetase Short Chain Family Member 2 (ACSS2), an enzyme converting acetate to acetyl-CoA, is known to regulate gene transcription and cellular processes through its nuclear localization under metabolic stress. However, its role in IBD remains unclear. In this study, immunohistochemistry of colon samples from UC patients revealed significantly elevated expression and nuclear localization of ACSS2 in inflamed tissues compared to non-inflamed regions, suggesting its involvement in IBD pathogenesis. Using Caco-2 and HT-29 intestinal epithelial cell lines stimulated with Tumor Necrosis Factor-α (TNF-α) or Dextran Sodium Sulfate (DSS) as in vitro models of inflammation, we observed upregulation of ACSS2 protein expression. ACSS2 inhibition significantly reduced the expression of pro-inflammatory cytokines such as Interleukin 8 (IL-8), IL-1β, and TNF-α, and attenuated activation of NF-κB and MAPK signaling pathways, as evidenced by decreased phosphorylation of p65 and ERK/MEK. These findings highlight a pro-inflammatory role for ACSS2 in IBD and identify it as a potential therapeutic target, warranting further validation in advanced models, including patient-derived organoids and in vivo systems.