Doxorubicin　(DOX)　is　a　potent　and　clinically　approved　chemotherapeutic　agent　for　cancers.　Although　human　serum　albumin　(HSA)　has　been　frequently　used　as　a　promising　drug　vehicle　for　DOX　to　reduce　its　doses　and　toxicities,　the　pH-dependent　binding　mechanism　of　HSA　with　DOX　is　still　poorly　understood.　This　hampers　the　rational　design　of　smart　HSA-based　cancer　microenvironment-responsive　DOX　delivery　systems　for　clinical　applications.　Here,　the　molecular　interactions　of　DOX　with　HSA　at　various　pH　levels　were　systematically　investigated　using　spectroscopic　and　in　silico　techniques.　Our　experiments　showed　that　the　binding　of　DOX　to　HSA　was　governed　by　static　quenching　mechanism　and　highly　affected　by　environmental　pH.　The　change　of　pH　altered　the　conformational　states　of　HSA,　which　may　regulate　the　binding　of　DOX　onto　HSA.　In　agreement　with　experimental　observations,　1000　ns-long　constant-pH　molecular　dynamics　simulations　elucidated　the　structural　basis　of　DOX　bound　to　HSA　at　different　pH.　The　computational　results　revealed　that　the　favored　binding　of　DOX　at　slight　alkaline　pH　may　be　attributed　to　the　intact　structure　of　HSA　and　increased　intermolecular　hydrogen　bonding.　In　contrast,　at　acidic　conditions,　the　extension　of　HSA　global　conformation　and　drop　of　local　structural　identity　reduced　the　number　of　intermolecular　hydrogen　bonds.　Our　study　provided　novel　structural　insights　into　the　molecular　mechanism　of　pH-dependent　interactions　of　HSA　with　DOX,　which　may　be　helpful　for　the　design　of　novel　HSA-based　microenvironment-responsive　drug　delivery　systems　for　anticancer　therapeutic　applications.　©　2021　Elsevier　B.V.