Two recently identified missense mutations (p. L84F and p. I107T) in GUCA1A, the gene coding for guanylate cyclase (GC)-activating protein 1 (GCAP1), lead to a phenotype ascribable to cone, cone-rod and macular dystrophies. Here, we present a thorough biochemical and biophysical characterization of the mutant proteins and their distinct molecular features. I107T-GCAP1 has nearly wild-type-like protein secondary and tertiary structures, and binds Ca2+ with a >10-fold lower affinity than the wild-type. On the contrary, L84F-GCAP1 displays altered tertiary structure in both GC-activating and inhibiting states, and a wild type-like apparent affinity for Ca2+. The latter mutant also shows a significantly high affinity for Mg2+, which might be important for stabilizing the GC-activating state and inducing a cooperative mechanism for the binding of Ca2+, so far not been observed in other GCAP1 variants. Moreover, the thermal stability of L84F-GCAP1 is particularly high in the Ca2+-bound, GC-inhibiting state. Molecular dynamics simulations suggest that such enhanced stability arises from a deeper burial of the myristoyl moiety within the EF1–EF2 domain. The simulations also support an allosteric mechanism connecting the myristoyl moiety to the highest-affinity Ca2+ binding site EF3. In spite of their remarkably distinct molecular features, both mutants cause constitutive activation of the target GC at physiological Ca2+. We conclude that the similar aberrant regulation of the target enzyme results from a similar perturbation of the GCAP1–GC interaction, which may eventually cause dysregulation of both Ca2+ and cyclic GMP homeostasis and result in retinal degeneration.