Abstract: Enolase 1 (ENO1) is a rate limiting enzyme of glycolysis, and participates in a variety of physiological and pathological processes according to its cell localization. In this study, zebrafish embryonic fibroblasts (PAC2 cell line) were used to study biochemical functions of ENO1 polypeptide and its covalent modifications. At first, methylated, acetylated, phosphorylated and unmodified eno1 peptides were artificially synthesized, and separately transduced into PAC2 cells. Then the treated cells were collected and examined by using CCK-8 assay, intracellular and extracellular lactate dehydrogenase (LDH) assay, mitochondrial and lysosomal integrity detection kits, as well as quantitative PCR Array. The results showed that the four polypeptides differentially changes cell proliferation, morphology of lysosomes and mitochondria, and glucose metabolic pathways. Furthermore, RNA-sequencing were conducted for the cells treated by acetylated Eno1 polypeptide and vehicle control Transcriptomal analysis indicated that acetylation modified Eno1 polypeptide significantly changed metabolism profile of sugar, lipid and protein, and is closely associated with carcinogenesis, viral infection and cardiomyopathy. These results suggest that multifaceted functional diversity of Enolase 1 may result from its various post-translational modifications.