Reticulon 3 (RTN3) is a neuronally-expressed reticulon family protein that was previously shown to negatively regulate BACE1, a protease that is required for the generation of β-amyloid peptides (Aβ) from amyloid precursor protein. Despite biochemical and morphological evidence that supports a role of RTN3 in the formation of neuritic amyloid plaques, no systematic analyses of RTN3 mutations in patients with Alzheimer's disease (AD) have yet been reported. RTN3 were targeted sequenced in 154 sporadic early-onset and 285 late-onset AD patients. Luciferase reporter assay and kymographs were performed to analysis the expression of RNT3 and BACE1-RFP particle mobility on cells transfected with wild-type or variants RTN3 constructs. We identified heterozygous variants such as c.-8G > T, c.17C > A, c.42C > T, and c.116C > T from patients in the early-onset AD group and c.-8G > T, c.17C > A, from patients in the late-onset AD group. Such variants of RTN3 were not observed in control individuals. Further biochemical studies show that the RTN3 c.-8G > T variant in the 5'-untranslated region appears to cause reduced expression of RTN3. The RTN3 c.116 C > T variant causes a change of codon T39 to M39 (T39 M). Overexpression of RTN3 T39 M in cultured neurons led to impaired axonal transport of BACE1. The variants found in this study are likely genetic modifiers for RTN3-mediated formation of neuritic plaques in AD.