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Ribosomal protein L34 antisense RNA 1 (RPL34-AS1) is novel long non-coding RNA, and was found to be down-regulated in colorectal cancer and gastric cancer. The role of RPL34-AS1 was still unknown in esophageal cancer. In our study, we found RPL34-AS1 expression levels in esophageal cancer tissue and specimens and cell lines were lower than in adjacent normal tissue specimens and normal esophageal cell line, respectively. In addition, esophageal cancer patients with Ⅲ-Ⅳ, T3-T4 or positive lymph node metastasis had lower RPL34-AS1expression levels than esophageal cancer patients with Ⅰ-Ⅱ, T1-T2 or negative lymph node metastasis, respectively. In survival analysis suggested esophageal cancer patients with low RPL34-AS1 expression had short overall survival than those with high RPL34-AS1 expression. Our in vitro experiments suggested RPL34-AS1 inhibits esophageal cancer cell proliferation, migration and invasion through down-regulating RPL34 expression. In conclusion, RPL34-AS1 functions as tumor suppressor in esophageal cancer.