The present study aimed to investigate the expression of LRP16 in the development of ESCC and the relationship between Hippo signaling pathway and LRP16. Immunohistochemistry was used to detect the expression of LRP16 in ESCC tissues. After transfection, the expression of LRP16 was detected by reverse transcription quantitative PCR (RT-qPCR) and western blot techniques. Cell counting kit (CCK-8), clone formation experiment, flow cytometry and wound healing were used to determine the proliferation, apoptosis, cell cycle and migration of ESCC cells. The changes of factors related to Hippo signaling pathway were determined via RT-qPCR and western blot experiments. The results showed that the LRP16 expression in ESCC tissues was higher than that in normal tissues. High expression of LRP16 was related to the depth of invasion, TNM stage and lymph node metastasis of ESCC. Furthermore, the knockdown of LRP16 inhibited proliferation, migration and promoted cell apoptosis and made cells arrested in G2/M phase. It also resulted in decreased expression of Yes-associated protein (YAP), and increased expression of mammalian STE20-like protein kinase (MST1/2), suggesting that LRP16 promoted the development of ESCC through Hippo signaling pathway. The results of this study suggest that LRP16 may be a carcinogenic gene of ESCC and promotes the progression of ESCC through the regulation of Hippo signaling pathway. Our study provides a new idea for the diagnosis and treatment of ESCC in the future.