Objective：This study aims to explore the molecular mechanism of ubiquitin-specific peptidase 14（USP14） instabilizing oxysterol binding protein-like 2（OSBPL2） expression by regulating the ubiquitination level of OSBPL2. Methods：HeLa cells was used as tool cells to detect the expression of OSBPL2 by overexpression，knockdown and activity inhibition of USP14. The regulation of USP14 on ubiquitin signal and ubiquitination level of OSBPL2 was investigated in vitro. The specific domain of USP14 interacting with OSBPL2，the interactional role of different domains and the key ubiquitination sites of OSBPL2 were determined by Co-immunoprecipitation（Co-IP）. Results：USP14 interacted with OSBPL2 and stabilized the expression of OSBPL2 without changing its transcriptional level. In vitro ubiquitination experiment and Co-IP showed that the catalytic（CAT） domain of USP14 interacted with the OSBP-related domain（ORD）of OSBPL2，which reduced the ubiquitination level of OSBPL2. The ubiquitin-like（UBL）domain of USP14 could promote the interaction between USP14 and OSBPL2. In addition，Lys209 and Lys361 could be the key sites for OSBPL2 ubiquitination and deubiquitination. Conclusion：USP14 modulated the ubiquitination level of OSBPL2 by regulating the ubiquitination of Lys209 and Lys361 and further stabilized OSBPL2 expression.