Recently, enolase 1 (ENO1) attracted scientists’ attention because it was identified as a glycolytic enzyme and an oncogene in carcinogenesis. However, downstream molecular events of ENO1 are poorly understood. It also remains unknown on the role of ENO1 in thyroid cancer (TC). Based on The Cancer Genome Atlas database (TCGA), we found that ENO1 was overexpressed in TC tissues. Knockdown of ENO1 inhibited and overexpression of ENO1 promoted the proliferation, colony growth and migration of TC cells. ENO1 also accelerated cell cycle progression and suppressed apoptosis in TC cells. RNA sequence data showed that numerous genes were regulated by ENO1. Gene Set Enrichment Analysis showed that ENO1 silencing inactivated signaling pathways of G2M Checkpoint and IL6/JAK/STAT3. In addition, cystatin 1 (CST1) and cystatin 4 (CST4) were downregulated by ENO1 knockdown in TC cells. Positive correlation between ENO1 and CST1 or CST4 was observed in TC tissues based on TCGA database. Furthermore, we also explored the function of CST1 and CST4 and found that downregulation of CST1 or CST4 blocked the proliferation and migration capacity of TC cells. In the future, we will investigate the mechanism by which ENO1 regulates CST1 and CST4. In addition, upstream regulator of ENO1 should be dissected in TC.