Acyl-CoA dehydrogenase short-chain (ACADS) is a crucial enzyme in the fatty acid metabolism pathway located in mitochondria. However, the expression level and prognostic value of ACADS in colorectal cancer (CRC) remain unclear. The mRNA and protein expression data of ACADS was obtained from The Cancer Genome Atlas (TCGA), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and Oncomine. Prognostic values of ACADS were calculated using Kaplan-Meier survival analysis. Correlations between ACADS and immune infiltration were estimated using TIMER, CIBERSORT, EPIC, quanTIseq, and xCell. The UALCAN and MEXPRESS databases were utilized for Methylation analysis. The co-expression analysis based on mRNA expression and interaction network of ACADS were performed via several online tools. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis on ACADS co-expressed genes were performed using the Metascape. The expression analysis demonstrated that ACADS was down-regulated in CRC tissues compared with paired normal tissue. Expression of ACADS was found to be significantly associated with clinical cancer stages and the consensus molecular subgroups (CMS) constituent ratio in CRC patients. Besides, lower ACADS expression was found to predict poor prognosis and be significantly associated with common immune checkpoint genes and MMR genes in CRC. ACADS expression levels were positively related to B cells, CD4 T cells, CD8 T cells, M1 macrophages, neutrophils, and Tregs, while negatively correlated with M0 macrophages, M2 macrophages. The methylation level of ACADS in normal tissues was significantly higher than that in tumor tissues, and several methylation sites were identified. The enrichment analysis suggested the co-expressed genes mainly enriched in cell mitochondrial metabolism. The present study provided multilevel evidences for expression of ACADS in CRC and the function of ACADS in prognostic prediction, immune infiltration, and methylation. ACADS might have the potential as the novel biomarker and therapeutic target in CRC patients.