Post-translational modifications (PTMs) play pivotal roles in controlling the stability and activity of the tumor suppressor p53 in response to distinct stressors. Here we report an unexpected finding of a short chain fatty acid modification of p53 in human cells. Crotonic acid (CA) treatment induces p53 crotonylation, but surprisingly reduces its protein, but not mRNA level, leading to inhibition of p53 activity in a dose dependent fashion. Surprisingly this crotonylation targets serine 46, instead of any predicted lysine residues, of p53, as detected in TCEP-probe labeled crotonylation and anti-crotonylated peptide antibody reaction assays. This is further confirmed by substitution of serine 46 with alanine, which abolishes p53 crotonylation in vitro and in cells. CA increases p53-dependent glycolytic activity, and augments cancer cell proliferation in response to metabolic or DNA damage stress. Since serine 46 is only found in human p53, our studies unveil an unconventional PTM unique for human p53, impairing its activity in response to CA. Because CA is likely produced by the gut microbiome, our results also predict that this type of PTM might play a role in early human colorectal neoplasia development by negating p53 activity without mutation of this tumor suppressor gene. (C) 2020 Elsevier Inc. All rights reserved.