Kruppel-like factors (KLFs) are transcription factors that control the expression of downstream genes. The role of KLFs has been reported in cancers. KLF16 promotes the proliferation of gastric cancer cells by upregulating p21, while suppresses the tumorigenesis of glioma through targeting TFAM. The function of KLF16 is controversial in cancer development. In this study, we aimed to investigate the role of KLF16 in retinoblastoma (RB). KLF16 was highly expressed in RB tissues and cells. Overexpression of KLF16 promoted the proliferation, growth and migration of RB cells. By contrast, KLF16 interference showed opposite effects. Cell cycle arrest and apoptosis were induced or repressed by KLF16 knockdown or overexpression, respectively. Mechanistically, BCL2 like 15 (BCL2L15), an apoptosis gene, was negatively regulated by KLF16. Luciferase reporter and ChIP assay showed that KLF16 transcriptionally repressed the expression of BCL2L15 by binding to its promoter. BCL2L15 was lowly expressed in RB tissues. Additionally, overexpression of BCL2L15 inhibited the proliferation and increased the apoptosis in RB cells. Our study identifies that KLF16 contributes to RB cell proliferation and migration by negatively regulating BCL2L15. (c) 2020 Elsevier Inc. All rights reserved.