Nonviral vectors present considerable advantages over viral counterparts in gene transfer. However, the poor expression efficiency of the transfected genes poses a challenge for their use in gene therapy, primarily due to the inability of these vectors to overcome various barriers, including the biological barriers. Here, we report that ZNF511-PRAP1 may be involved in the recognition and inactivation of transfected plasmids. ZNF511-PRAP1 is induced by transfection of plasmid DNA and suppresses the transcription of transfected plasmids. It binds directly to the p21 promoter in transfected plasmids but not the endogenous counterpart. Similarly, ZNF511-PRAP1 suppresses the expression of the green fluorescent protein reporter gene on transiently transfected plasmids but not an integrated red fluorescence reporter gene with the same cytomegalovirus (CMV) promoter. Therefore, ZNF511-PRAP1 is able to differentiate between exogenous/nonintegrated and endogenous/integrated DNA. The suppression by ZNF511-PRAP1 is independent of DNA methylation and can be abolished by trichostatin A (TSA) treatment and knockdown of HDAC2 and/or ZNF511-PRAP1. Furthermore, ZNF511-PRAP1 interacts directly with HDAC2. Our results revealed that transfected plasmids are recognized by ZNF511-PRAP1 and suppressed by a repressor complex comprising ZNF511-PRAP1 and HDAC2 and suggest that ZNF511-PRAP1 could play a role as a potential molecular barrier in nonviral transgene expression.