Previous studies have documented mitochondrial dysfunction during the critical node (CN) of rice (Oryza sativa) seed aging, including a decrease in the capacity of NADH dependent O2 consumption. This raises the hypothesis that changes in the activity of NADH:ubiquinone oxidoreductase (complex I) may play a role in seed aging. The composition and activity of complex I was investigated at the CN of aged rice seeds. Using BN-PAGE and SWATH-MS 52 complex I subunits were identified, nineteen for the first time to be experimentally detected in rice. The subunits of the matrix arm (N and Q modules) were reduced in abundance at the CN, in accordance with a reduction in the capacity to oxidise NADH, reducing substrate oxidation and increase ROS accumulation. In contrast, subunits in the P module increased in abundance that contains many mitochondrial encoded subunits. It is proposed that the changes in complex I abundance subunits may indicate a premature re-activation of mitochondrial biogenesis, as evidenced by the increase in mitochondrial encoded subunits. This premature activation of mitochondrial biogenesis may under-pin the decreased viability of aged seeds, as mitochondrial biogenesis is a crucial event in germination to drive growth before autotrophic growth of the seedling is established.