Objective To investigate the influence of the N-terminal mutations of Annexin A11 (ANXA11) protein, p.G38R and p.D40G, on the subcellular localization of this protein and their mechanism of action in inducing amyotrophic lateral sclerosis (ALS).Methods Eukaryotic expression vectors which expressed ANXA11 wild-type protein, ANXA11 p.G38R protein, or ANXA11 p.D40G protein were constructed, and a laser scanning confocal microscope was used to investigate the subcellular localization of these three proteins in HEK293 cells.Results ANXA11 wild-type protein, ANXA11 p.G38R protein, and ANXA11 p.D40G protein formed a vesicular structure in HEK293 cells, and the p.G38R group had significantly larger vesicles than the ANXA11 wild-type group (P=0.002) and the p.D40G group (P=0.006), while there was no significant difference between the latter two groups (P=0.791). In a single cell, the ANXA11 wild-type group had significantly more vesicles than the p.G38R group (P=0.002) and the p.D40G group (P<0.001), and there was no significant difference between the p.G38R group and the p.D40G group (P=0.516). Compared with ANXA11 wild-type protein, p.G38R protein and p.D40G protein had significantly reduced distribution in the nucleus (both P<0.001), and there was no significant difference between p.G38R protein and p.D40G protein (P=0.519).Conclusions The p.G38R and p.D40G mutations reduce the number of vesicles of ANXA11 protein and the distribution of ANXA11 protein in the nucleus, which may be one of the mechanism of ALS.