Background Circular RNA (circRNA), an endogenous non-coding RNA (ncRNA), plays a critical role in gene regulation and shows potential as diagnostic or prognostic biomarkers of human diseases. However, to date there has been little research on the role of circRNAs in the occurrence and development of esophageal cancer. Methods We adopted a three-stage design first using a high-throughput microarray chip to screen for differentially expressed circRNAs in esophageal squamous cell carcinoma (ESCC), then performing a validation study via quantitative reverse transcription polymerase chain reaction (qRT-PCR), and followed by functional analysis using in vitro experiments. In the discovery stage, we screened seven paired samples of ESCC and corresponding adjacent normal-appearing tissues for differentially expressed circRNAs. In the validation stage, we compared the expression of candidate circRNAs in 45 patients with ESCC and 45 healthy controls. In the functional analysis stage, we used three types of cell lines to explore the function of hsa_circ_0030162. Results In the discovery stage, we observed 459 up-regulated and 275 down-regulated differentially expressed circRNAs with FC ≥2.0 and P <0.05. Among these, hsa_circ_0030162 was ranked as the top down-regulated circRNA (FC=12.1). Thus, we selected this circRNA for validation using qRT-PCR and compared its expression in 45 patients with ESCC and 45 healthy controls. If using tissue hsa_circ_0030162 as the diagnostic biomarker, the AUC was 0.743 (95% CI: 0.639-0.848) with a sensitivity of 73.33% and a specificity of 66.67%. If using plasma hsa_circ_0030162 as the diagnostic biomarker, the AUC was 0.735 (95% CI: 0.632-0.837) with a sensitivity of 73.33% and a specificity of 62.22%. The in vitro experiments revealed that hsa_circ_0030162 could promote apoptosis and inhibit proliferation, migration, and invasion of ESCC cells. Luciferase reporter assays proved that it could bind to miR-125a-3p, a non-coding RNA involved in carcinogenesis. Conclusions Our results provide the first evidence that hsa_circ_0030162 plays an important role in the development and progression of ESCC. In addition, we illustrate the diverse roles of hsa_circ_0030162 in regulating cell proliferation, migration, invasion and apoptosis. Based upon our findings, we propose that hsa_circ_0030162 may act as a promising biomarker for clinical diagnosis of ESCC and suggest a new pathway for the development of anti-tumor therapeutics. Funding: The present study was supported by the National Natural Science Foundation of China [81673249], National Key R&361 D Program of China [2017YFC0907000], Social Development Project in Jiangsu Province [BE2015694], Scientific Research Innovation Project for Graduate Students in Jiangsu Province [KYCX17_1293], and Priority Academic Program Development of Jiangsu Higher Education Institutions PAAD]. Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: This study was approved by the Ethics Committee of Nanjing Medical University. After informed consents from all participants, questionnaires were used to collect demographic data.