In this work, we report on photophysical studies of the anticancer drug topotecan (TPT) in aqueous solutions at different pHs. We used steady-state (UV-visible absorption and emission) and time-resolved picosecond (ps) emission spectroscopies to investigate the role of the H-bonding interactions as well as the proton concentration (pH = 0.48-7.40) on the behavior of topotecan (TPT) in its ground- (S0) and electronically first (S1) excited-states. At physiological conditions (pH = 7.40), the drug exists at S0 in equilibrium between the enol (E), cation (C), and zwitterion (Z) forms. The photoformation of Z* (τZ = 5.83 ns) occurs from directly excited (λexc = 371 nm) E as the two-step reaction: E*→C*→Z*. In this process, a very fast (less than 10 ps) protonation of E* leads to C*, which subsequently undergoes fast (580 ps) deprotonation to give Z* as the final photoproduct. At higher proton concentrations (pH = 0.48-1.31), a ground-state equilibrium exists between three different cationic species (C1, C2, and C3). The proton motion from the acidic solution to the C forms of TPT to give the reactions C1*→C2*→C3* is governed by the proton diffusion. In these conditions, both dynamic and static quenching occurs. The rate constant values k*(DPT1) and k*(DPT2) for the direct protonation of C1* and C2*, respectively, depend on the pH of the surrounding. The number of protons implicated in the reaction changes from ∼2 (pH = 0.48-0.78) to ∼1 (pH = 0.78-1.31), thus indicating the existence of two different reactions and proton-transfer dynamics. These results evidence the conformational, structural, and dynamical changes of aqueous solutions of TPT with the pH of the environment. They should help to understand the molecular structure/activity of TPT at cellular level.