Objective  To explore the relationship between miR-26a and metadherin (MTDH), and to verify the relationship between miR-26a and MTDH in vivo in nude mice.    Methods  Immunohistochemical SP staining method  was used to detect the expression of MTDH and in situ hybridization was used to detect the expression of miR-26a in 86 cases of esophageal cancer, and the correlation between the expressions was analyzed. The bioinformatics prediction Targetscan Human 7.2 software could predicte the binding fragment of MTDH on the miR-26a sequence, and the luciferase report experiment was used to verify the targeted regulatory relationship between MTDH and miR-26a. Nude mice were injected esophageal cancer cell lines subcutaneously which were lentiviral interferenced and overexpressed miR-26a to observe the formation of tumors. The tumors from nude mice were made into paraffin. and each was detected. The expression of MTDH in miR-26a in the tumor groups was detected by immunohistochemical staining and in situ hybridization and the relationship was analyzed.    Results  The expression of miR-26a in esophageal cancer tissues was significantly lower than that in paired normal esophageal tissues, and the expression of MTDH in esophageal cancer tissues was significantly higher than that in paired normal esophageal tissues. The expression of miR-26a was related to the patient’s pathological grade (P<0.05), N stage (P<0.05), and tumor volume (P<0.01). The expression of MTDH in esophageal cancer was related to the N stage (P<0.05) and the degree of differentiation (P<0.01). Targetscan Human7.2 bioinformatics software predicted that the MTDH gene contained a target sequence of hsa-miR-26a.The luciferase reporter gene experiment also verified the targeted regulation relationship between miR-26a and MTDH. The expression of miR-26a was the highest in KYSE-450 cells and the lowest in Eca109 cells. The mRNA expression of MTDH in the lv-miR-26a group was significantly lower than that in the lv-NC group, and the mRNA expression in the lv-miR-26a-inhibitor group was significantly  higher than that in the lv-NC group. After tumor formation, miR-26a expression increased and MTDH expression decreased in miR-26a group. After tumor formation, the expression of miR-26a decreased and the expression of MTDH increased in miR-26a inhibitor group.    Conclusion  MiR-26a can inhibit the expression of MTDH in esophageal cancer cells. Both in vitro and in vivo experiments can verify the targeted regulatory relationship between miR-26a and MTDH. MiR-26a may play a role in the occurrence and development of esophageal cancer through the MTDH.