Amomum tsaoko is an important food and medicinal plant. Its dried fruit is rich in alkaloids and is known to have a notable laxative effect. Nevertheless, it remains unclear whether alkaloids from A. tsaoko can relieve constipation. The present study sought to purify alkaloids from A. tsaoko via the macroporous resins method and to investigate the laxative activity of alkaloid-rich A. tsaoko extract (ARAE). HP-20 was identified as the most efficient resin for purifying alkaloids from A. tsaoko. ARAE was predominantly concentrated in indole-3-carboxaldehyde, p-octopamine, phyllalbine, synephrine, lumichrome, and isatidine. The results of animal experiments demonstrated that ARAE markedly promoted gastrointestinal motility and shortened the defecation time of mice with functional constipation (FC). ARAE not only significantly affected the serum level of AChE but also upregulated the expression of 5-HT4R, c-Kit, SCF and smMLCK as well as the protein expression of c-Kit in the colon. Furthermore, ARAE increased the number of goblet cells in the colon, facilitated the secretion of acidic mucus, and increased the expression of intestinal barrier factors. Importantly, ARAE had a considerable influence on the structure and composition of the gut microbiota in mice with FC. The correlation analysis results suggested that ARAE may facilitate intestinal microecological balance by regulating key microbial populations, including Lachnospiraceae_NK4A136_group, Rikenellaceae_RC9_gut_group, norank_f_Desulfovibrionaceae, norank_o_Clostridia_vadinBB60_group, and Faecalibaculum. ARAE can alleviate FC by regulating gastrointestinal motility and the intestinal microecosystem. The potential applications of A. tsaoko and ARAE in the development of laxative health foods and pharmaceuticals are significant and warrant further investigation.