PIWIL2, called HILI in humans, is a member of the PIWI subfamily. This subfamily has highly conserved PAZ and Piwi domains and is implicated in several critical functions, including embryonic development, stem-cell self-renewal, RNA silencing, and translational control. However, the underlying molecular mechanism remains largely unknown. Transforming growth factor-β (TGF-β) is a secreted multifunctional protein that controls several developmental processes and the pathogenesis of many diseases. TGF-β signaling is activated by phosphorylation of transmembrane serine/threonine kinase receptors, TGF-β type II (TβRII), and type I (TβRI), which are stabilized by Hsp90 via specific interactions with this molecular chaperone. Here, we present evidence that HILI suppresses TGF-β signaling by physically associating with Hsp90 in human embryonic kidney cells (HEK-293). Our research shows that HILI mediates the loss of TGF-β-induced Smad2/3 phosphorylation. We also demonstrate that HILI interacts with Hsp90 to prevent formation of Hsp90-TβR heteromeric complexes, and improves ubiquitination and degradation of TβRs dependent on the ubiquitin E3 ligase Smurf2. This work reveals a critical negative regulation level of TGF-β signaling mediated by HILI (human PIWIL2) by its ability to interact with Hsp90 and promote TβR degradation.