Herein we report on the impact of C3'- and C4'-substitution on the photophysics of the heptamethine cyanine dye (Cy7) using electron-withdrawing (-CN) and donating (-OMe) groups. Frontier orbital analysis predicts that C3'-substitution mainly perturbs the HOMO, while C4'-substitution mainly perturbs the LUMO. Position- and substituent-dependent derivatization should thus enable tuning of the HOMO-LUMO gap, and consequently lead to selective red- or blue-shifting of the S₁ ← S₀ absorption band. Even though absorption spectra shift accordingly, fluorescence lifetimes deviate from simple energy-gap law predictions. However, within each substituent class, the more red-absorbing dye shows the shorter lifetime. Solvatochromic studies on the parent Cy7 dye further show that its fluorescence lifetime is independent of viscosity and is instead quenched by OH-containing solvents via an energy transfer mechanism. Transient absorption measurements covering fs to μs time scales further point to neither significant excited-state isomerization nor triplet formation. Cyclic voltammetry experiments show increasingly reversible reductions for C4'-substituted derivatives, likely due to the suppression of radical-radical dimerization pathways. Overall, these findings provide detailed insight into how solvent effects and regio-specific substitution modulate the photophysics and electrochemistry of Cy7, aiding in the rational design of near-infrared fluorophores.