Background: Long non-coding RNA (lncRNA) U731166 and microRNA (miR)-3607-3p are two ncRNAs with critical roles in cancer biology, while their involvement in esophageal squamous-cell carcinomas (ESCC) is unclear. We predicted that U731166 and miR-3607-3p might interact with each other. This study aimed to investigate their role and interaction in ESCC. Objectives: This study was therefore conducted to explore the involvement of U731166 and miR-3607-3p in ESCC, with a focus on the interaction between them. Materials and Methods: Paired ESCC and non-tumor tissue samples were recruited from 72 ESCC patients. By RTQpcr, level of U731166 and miR-3607-3p in paired tissues was measured. By RNA-RNA pulldown assay, the direct interaction between U731166 and miR-3607-3p was detected. U731166 overexpression or miR-3607-3p overexpression was performed to investigate their role in regulating the expression of each other. By RT-qPCR and Western blot analysis, the role of U731166 and miR-3607-3p in regulating the level of TGFBR1 was assessed. By Transwell assays, cell invasion and migration were analyzed. Results: Compared to non-tumor tissues, U731166 was highly upregulated in ESCC, while miR-3607-3p was downregulated in ESCC. U731166 and miR-3607-3p directly interacted with each other, but they are not closely correlated and did not regulate the level of each other. Moreover, U731166 reversed the role of miR-3607-3p in downregulating TGFBR1 and inhibiting cancer cell invasion and migration. U731166 and miR-3607-3p were closely associated with patients' tumor metastasis but not tumor size. Conclusion: U731166 may upregulate TGFBR1 by sponging miR-3607-3p in ESCC cells to promote tumor metastasis.