Probiotic supplementation has become a prominent method of decreasing ammonia emissions in poultry production. The present study was conducted to investigate the influence of Lactobacillus plantarum on ammonia emission, immune responses, antioxidant capacity, cecal microflora and short chain fatty acids, and serum metabolites in broilers challenged with ammonia. A total of 360 1-day-old yellow-feathered broilers were randomly divided into three treatment groups: birds fed with a basal diet (CON), a basal diet supplemented with ammonia (AN), and a basal diet supplemented with 2.5 × 108 CFU L. plantarum kg-1 and challenged with ammonia (LP). Data showed that L. plantarum supplementation decreased ammonia more than 30% from day 48, and significantly reduced the levels of serum urea nitrogen and ammonia, fecal urease, and ammonium nitrogen compared with those on CON. Compared with AN and CON treatments, LP administration increased (p < 0.05) the concentration of serum immunoglobulin Y (IgY), IgM, and IL-10, as well as the serum total-antioxidant capacity (T-AOC) and GSH-Px, and decreased (p < 0.05) IL-1β, IL-6, and TNF-α. Furthermore, birds supplemented with LP had higher (p < 0.05) cecal contents of short chain fatty acids (SCFAs) than AN birds and had more butyrate than CON birds. Data from 16s high throughput sequencing showed that LP supplementation significantly increased (p < 0.05) the Shannon and Simpson indices of bird cecal microflora, and Alloprevotella dominated the LP birds. The function prediction of cecal microflora indicated that LP treatment significantly increased alanine aspartate and glutamate metabolism, starch and sucrose metabolism, exosome, mismatch repair, homologous recombination, DNA repair and recombination proteins, and amino acid-related enzymes. The serum metabolome showed that LP supplementation significantly changed the aminoacyl-tRNA, pantothenate and acetyl-coenzyme A, arginine, phenylalanine, tyrosine and tryptophan, valine, leucine, and isoleucine biosynthesis; purine, beta-alanine, galactose, histidine, alanine, aspartate and glutamate, glyoxylate and dicarboxylate, pyruvate and thiamine metabolism, melanogenesis, and citrate cycle.