Insulators are noncoding gene regulatory elements in eukaryotic genome, which function as chromatin partitioning boundaries, and block interference across different chromatin domains. To facilitate modular construction of synthetic gene circuit that is usually composed of multiple transcription cassettes, unwanted cross-regulations between different cassettes should be avoided. Here, we developed a quantitative method to characterize the functional effect of three insulators on the cross-regulations of six promoters in mammalian cells. We showed that the unwanted cross-regulations displayed a threshold-like effect, and the threshold position varied along with the context of promoters and insulators. We tested the function of insulators in both cascade and sensory switch circuits assembled in episomal plasmid vectors, and showed that the insulation effect was mainly revealed on the first regulatory layer of the cascade circuit. A deviation on the response curve of the sensory switch circuit with or without insulators was observed, but response intensity of some sensory switch circuits were not affected. Therefore, our results provided a general guide on the selection of insulators with varying promoters in episomal synthetic gene circuits in mammalian cells, which may be useful to reduce the effect of the unwanted cross-regulations.