Objective Inhibitors of cyclin‐dependent kinase 4 and 6 (CDK4/6) are targeted therapeutic drugs for breast cancer treatment. The mechanism of resistance to these inhibitors requires further investigation. Methods We used bioinformatics to screen differentially expressed genes between cells that were susceptible and resistant to CDK4/6 inhibitors. Quantitative real‐time PCR (qRT‐PCR) was used to identify gene expressions in different cell lines. Cell viability, colony formation, cell cycle, and apoptosis assays were used to evaluate the effect of carboxypeptidase vitellogenic like (CPVL) on breast cancer cells under the condition of CDK4/6 inhibitors. Gene set enrichment analysis (GSEA) suggested the potential regulatory pathway of CPVL in breast cancer. Xenograft formation assay was conducted in nude mice to study the role of CPVL in vivo. Results Based on bioinformatics analysis and qRT‐PCR, CPVL was identified more abundantly in cells that were resistant than sensitive to CDK4/6 inhibitors. Overexpressed or knocked down CPVL regulated the effects of CDK4/6 inhibitors in resistant cell lines. GSEA showed that resistance might be induced by CPVL through altered phosphatase and tensin homolog (PTEN)‐related pathways. Our findings showed that CPVL negatively regulates PTEN to impact the anticancer effects of CDK4/6 inhibitors in vitro and in vivo. Conclusion CPVL might be a key factor in regulating breast cancer resistance to CDK4/6 inhibitors.