The aim of this study was to clarify the importance of microRNA‑375 (miR‑375) expression in patients with esophageal squamous cell carcinoma (ESCC) and to examine the in vivo antitumor effects of miR‑375 in a model of ESCC using a non‑viral delivery system. We estimated the miR‑375 and LDHB and AEG‑1/MTDH mRNA expression of the ESCC tumors from 85 patients. The correlation between the miR‑375 expression and clinicopathological features, including the prognosis, were evaluated. The presence of high miR‑375 expression was associated with lymphatic vessel invasion, while a low expression of miR‑375 significantly correlated with a poor prognosis for the 85 ESCC patients. We also found that there was a significant inverse correlation between the expression of miR‑375 and that of LDHB. Before the examination of miR‑375 in the in vivo assay, we confirmed that atelocollagen prolonged the accumulation of miRNA by using fluorescently‑labeled miRNA and an in vivo imaging system. We injected the miR‑375/atelocollagen complex or a control‑miRNA/atelocollagen complex into mice bearing TE2 and T.Tn xenografts via subcutaneous (s.c.) injections. The growth of both the TE2 and T.Tn tumors in the miR‑375 groups was significantly suppressed compared with that in the control‑miRNA groups. In addition, The LDHB mRNA expression of TE2 xenografts was significantly downregulated after miR‑375 treatment. In conclusion, it might be possible for the level of miR‑375 expression to be a utilized as a prognostic indicator for ESCC patients. The administration of miR‑375 using a non‑viral delivery might represent a powerful new treatment for ESCC.