Purpose <p>Chemotherapy remains a key treatment option for cancer therapy. Epirubicin (EPI) is an important antitumor agent. However, the short half-life and pronounced toxic side effects have resulted in suboptimal clinical outcomes. To overcome these challenges, we employed a prodrug strategy combined with bio-nanotechnology to achieve effective treatment with satisfactory safety. In addition, we investigated how the position of disulfide bond affects drug release, pharmacokinetics, and antitumor efficacy of EPI, while also analyzing the underlying mechanisms.</p> Methods <p>We designed and synthesized three lipophilic EPI-C₁₈ prodrugs (α-ESC, β-ESC, γ-ESC), which were then formulated into carrier-free nanoassemblies (α-ESC NAs, β-ESC NAs, γ-ESC NAs). In vitro studies were conducted to evaluate the stability and drug release of these nanoassemblies, followed by in vivo pharmacokinetic and antitumor efficacy assessments, providing valuable insights into their therapeutic potential.</p> Results <p>The placement of the disulfide bond plays a critical role in influencing the stability, drug release, cytotoxicity, pharmacokinetics, and antitumor effectiveness of the nanoassemblies. Specifically, β-ESC NAs displayed the strongest antitumor activity.</p> Conclusion <p>By constructing reduction-responsive EPI prodrug nanoassemblies, we achieved a highly effective and low-toxicity therapeutic outcome. β-ESC NAs proved to be the most optimal EPI prodrug nanoassembly. This study provides a promising avenue for the clinical use of chemotherapy agents.</p>