The Cell wall-associated kinase family have annotated approximately 130 WAK genes in the genome of rice (Oryza sativa), which play an important role in rice growth and development and stress responses. Here, we investigated the regulation and physiological mechanism of OsWAK16, an encoding gene of the cell wall-associated kinase WAK16-RLK, on rice seed vigor and anti-aging ability. The results showed that before and after artificial aging, the seed vigor of OsWAK16 mutants and overexpression lines was significantly lower and higher than that of wild-type seeds, respectively, indicating that OsWAK16 positively regulates the anti-aging ability of seeds. Physiological and biochemical analyses indicated that compared with wild-type seeds before and after artificial aging treatment, malondialdehyde (MDA) content and electrical conductivity (EC) of seed soaking solution of OsWAK16 mutant seeds were significantly increased, while antioxidant enzyme activity was significantly decreased. The reverse was true in overexpression seeds. In addition, the differential expression of OsWAK16 in three types of seeds, whether artificially aged or not, also caused synergistic changes in the expression of other seed vigor-related genes OsPER1A、OsbZIP23、OsPIMT1、OsSdr4、OsMSRB5 and OsHSP18.2 etc. Therefore, it is speculated that OsWAK16 may work synergistically with other seed vigor-related genes to clear reactive oxygen species in cells, thereby regulating seed vigor and anti-aging capacity.