Abstract Receptor tyrosine kinases (RTKs) play a crucial role in the regulation of intracellular signal transduction, underscoring their significance as targets for drug therapy. Despite the widespread clinical use of kinase inhibitors, the increasing occurrence of off-target effects and drug resistance makes it urgent to explore alternative approaches to modulate RTKs functions. Here, we propose an approach for attenuating cell-surface receptor signaling, termed Aptamer-directed Phosphatase Recruiting Chimeras (Apt-PRCs). The Apt-PRC is composed of an aptamer to recruit phosphatases and a binder to target receptors. As a proof-of-concept, we design and construct Apt-PRCs intended for direct dephosphorylation of tyrosine residues on the receptor targets, i.e., epidermal growth factor receptor and mesenchymal-epithelial transition factor, respectively. The as-developed Apt-PRCs manage to inhibit specifically and efficiently the reception and transmission of phosphorylation signals both in vitro and in vivo. Furthermore, it is discovered that the induced dephosphorylation could enhance the susceptibility to gefitinib in drug-resistant cancer cells and a xenograft mouse model, indicating the potential of Apt-PRCs to overcome drug resistance in cancer. This work offers a versatile methodology to design molecular mediators to modulate receptor phosphorylation so as to regulate the downstream signal transduction and overcome drug resistance.