Previously we identified CACNA1I, encoding CaV3.3, as a disease gene for neurodevelopmental disorders and epilepsy. Here we examine two further de novo missense variants in the CACNA1I gene, affecting the same residue located in the activation gate at the cytoplasmic end of the IS6 helix. A398E was identified in a patient with seizures, severe intellectual disability, hypotonia and developmental delay, while the patient with the A398V variant presented with microcephaly and mild to moderate developmental delay but no seizures. We analyzed current properties using whole-cell patch-clamp recording in tsA201 cells and examined the effects of the amino acid substitutions in our CaV3.3 structure model. A398E showed no effect on current density, but significantly left-shifted voltage-dependence of activation and inactivation, and substantial slowed activation, deactivation, and inactivation. In contrast, A398V caused a reduction of current density, a mild shift of activation and little to no changes in the gating kinetics. Structure modeling indicated that the two substitutions differentially affect stabilization of the different gating states of CaV3.3. Thus, gain-of-function effects of A398E are in line with the severe patient phenotype. The increased calcium influx at rest and after action potentials, due to the increased window currents and slowed kinetics, likely leads to calcium toxicity in developing neurons. Thus, altered differentiation and cell death might explain the neurodevelopmental defects. Additionally, hyperpolarized activation could cause increased excitability, explaining the seizures. The effects of A398V are much smaller and correspond to the mild patient phenotype. The absence of severely altered gating seemingly protected the patient from developing seizures. This corroborates the link between CaV3.3 and neurodevelopmental disorders, and underlines that not solely the position, but also the type of substitution determines the severity and nature of the disease.