Acyl-CoA oxidase 2 (Acox2) is an enzyme involved in peroxisomal bile acid synthesis and branched-chain fatty acid degradation. Acox2 knockout (−/−) mice spontaneously developed liver cancer with marked lymphocytic infiltrate. Tandem-affinity purification coupled with mass spectrometry analysis revealed that Acox2 interacted with methylcrotonoyl-CoA carboxylase followed by co-immunoprecipitation confirmation. Here we reported that non-histone lysine crotonylation (Kcr) levels were downregulated in Acox2−/− mice livers. Interestingly, Kcr signals were concentrated in the nucleus of tumor cells but mostly located in the cytoplasm of adjacent normal liver cells of Acox2−/− mice. Quantitative analysis of the global crotonylome further revealed that 54% (27/50) of downregulated non-histone Kcr sites were located in mitochondrial (11/50) and peroxisomal (17/50) enzymes including Ehhadh, Scp2, Hsd17b4, Crot, Etfa, Cpt1a, Eci1/2, Hadha, Etfdh, and Idh2. Subsequent site-directed mutagenesis and transcriptome analysis revealed that Ehhadh K572cr might have site-specific regulatory roles by downregulating TOP3B expression that lead to increased DNA damage in vitro. Our findings suggested Acox2 is a regulator of Kcr that might play critical role on hepatic metabolic homeostasis.