Slurry acidification has been shown to be effective in reducing environmentally damaging gases. However, this involved the use of concentrated acids on farms. Therefore, due to the health and safety concerns, there is an interest in self-acidification of slurry technique. This study was designed to determine the microbial dynamics leading to self-acidification of slurry. A fresh cattle slurry was amended 10% brewing sugar and stored over 30 days. This fermentable carbon source promoted self-acidification of the slurry from pH 7.0 to 4.7 within four days, and was associated with the accumulation of lactic acid and a reduction in methane and relative ammonia emissions. A metagenomics approach through next generation sequencing (NGS) using an Illumina MiSeq platform was used to determine the microbial diversity and dynamics (bacteria and archaea) in the stored amended slurry. 16S ribosomal ribonucleic acid (rRNA) sequence data revealed the presence of the Order of Lactobacillales was associated with the lactic acid production. The operational taxonomic units (OTUs) abundance indicates that the methanogenic community was dominated by hydrogenotrophic methanogens from the member Order of Methanobacteriales, Methanomicrobiales, and Methanosarcinales. The decrease in tolerance by the methanogens in the self-acidified slurry was probably the main reason for the reduced methane emission. These results confirm, at the microbial level, the mechanism of inhibiting methane production via self-acidification during storage period.