Lysine crotonylation (Kcr) is a recently identified post‐translational modification (PTM) that is regulated by an acetyltransferase, p300. The p300‐catalyzed histone Kcr is able to stimulate transcription to a greater degree than the well‐studied histone lysine acetylation (Kac). Despite these progresses, the global Kcr substrates regulated by p300 remain largely unknown, hindering efforts to establish mechanistic links between Kcr and p300‐mediated phenotypes. Here, a quantitative proteomics study to characterize the p300‐regulated lysine crotonylome is reported. A total of 816 unique endogenous crotonylation sites are identified across 392 proteins, with 88 sites from 69 proteins being decreased by more than 0.7‐fold (log2 < 0.5) and 31 sites from 17 proteins being increased by more than 1.4‐fold (log2 > 0.5) in response to p300 knockout (KO). The most downregulated crotonylome alterations under p300 deficiency concern components of the nonsense‐mediated decay, infectious disease, and viral/eukaryotic translation pathways. Moreover, some p300‐targeted Kcr substrates are potentially linked to diseases such as cancer. Taken together, this study reveals the lysine crotonylome in response to p300, which sheds light on the role for lysine crotonylation in regulation of diverse cellular processes and provides new insights into mechanisms of p300 functions.