Sodium butyrate is a commonly used feed additive and can reduce ammonia (NH) emissions from laying hens, but the mechanism of this effect is unknown. In this study, the sodium butyrate and cecal content of Lohmann pink laying hens were measured, and in vitro fermentation experiments and NH-producing bacteria coculture experiments were carried out to explore the relationship between NH emissions and its associated microbiota metabolism. Sodium butyrate was found to significantly reduce NH emission from the cecal microbial fermentation of Lohmann pink laying hens (P < 0.05). The concentration of NO-N in the fermentation broth of the sodium butyrate-supplemented group increased significantly, and the concentration of NH-N decreased significantly (P < 0.05). Moreover, sodium butyrate significantly reduced the abundance of harmful bacteria and increased the abundance of beneficial bacteria in the cecum. The culturable NH-producing bacteria consisted mainly of Escherichia and Shigella, such as Escherichia fergusonii, Escherichia marmotae and Shigella flexnerii. Among them, E. fergusonii had the highest potential for NH production. The coculture experiment showed that sodium butyrate can significantly downregulate the expression of the lpdA, sdaA, gcvP, gcvH and gcvT genes of E. fergusonii (P < 0.05), thus reducing the NH emission produced by the bacteria during metabolism. In general, sodium butyrate regulated NH-producing bacteria to reduce NH production in the cecum of laying hens. These results are of great significance for NH emission reduction in the layer breeding industry and for future research.