Loss of function studies played a critical role in dissecting gene function and downstream regulation networks. However, the most used knockout approaches, including CRISPR/Cas9, Cre/LoxP, shRNAs, and more, require days to achieve protein depletion. This will lead to the concern of the secondary effect of gene function, and cellular phenotype associate transcriptome changes are likely to bias the molecular mechanism study. To mitigate this problem, the auxin-inducible degron (AID) system was developed as an appealing approach for targeted cell protein degradation. Upon auxin treatment, ectopically expressed OsTIR1 adaptor protein connects cellular SCF E3 complex and AID-fused protein as a tertiary complex to trigger proteosome-mediated protein degradation. Although this system was applied to cell and animal models, the long-term auxin treatment and degradation resistance effect has never been reported yet. In this study, we derived multiple miniAID-protein knockin cell lines and a Ctcf-miniAID knockin mouse strain to systematically determine the degradation resistance mechanisms. Compared to other control protein-AID systems, CTCF-miniAID frequently acquired auxin treatment resistance in both cell lines and knockin mouse-derived primary cells. We revealed the three major resistance mechanisms, including nonsense mutations of the CTCF coding sequence to truncate miniAID peptide, a missense point mutation at the minAID coding region to dissociate the E3 complex targeting and epigenetic silence of OsTIR1 adaptor proteins. In summary, our innovative study will significantly extend our understanding of the AID system and promote future applications with cautious design.