Seed vigor is a crucial trait for the direct seeding of rice.  Here we examined the genetic regulation of seed vigor traits in rice, including germination index (GI) and germination potential (GP), using a genome-wide association study approach.  One major quantitative trait locus, qGI6/qGP6, was identified simultaneously for both GI and GP.  The candidate gene encoding the cytochrome c oxidase subunit 5B (OsCOX5B) was validated for qGI6/qGP6.  The disruption of OsCOX5B caused the vigor traits to be significantly lower in Oscox5b mutants than in the japonica Nipponbare wild type (WT).  Gene co-expression analysis revealed that OsCOX5B influences seed vigor mainly by modulating the tricarboxylic acid cycle process.  The glucose levels were significantly higher while the pyruvic acid and adenosine triphosphate levels were significantly lower in Oscox5b mutants than in WT during seed germination.  The elite haplotype of OsCOX5B facilitates seed vigor by increasing its expression during seed germination.  Thus, we propose that OsCOX5B is a potential target for the breeding of rice varieties with enhanced seed vigor for direct seeding.