Fermented dairy food, such as yogurt, exhibits some beneficial effects on consumers, including alleviating constipation. In this study, Lactobacillus delbrueckii subsp. bulgaricus DPUL-36, Lactobacillus paracasei DPUL-40 and Lactobacillus paracasei DPUL-44 were used as combined starter cultures at a bacterial cell ratio of 1 : 1 : 1 for reconstituted skim milk fermentation. The milk fermented with the combined starter culture showed good sensory properties. During the storage period, the yogurt showed high lactic acid bacteria vitality and quality stability. Constipated BALB/c mice induced by loperamide (Lop) were orally administered with the combined starter culture fermented milk for 14 days. The results clearly showed that oral administration of the fermented milk relieved Lop-induced constipation in the mice, as evidenced by the significantly increased fecal water content, reduced first black stool time, improved gastrointestinal transmission rate, recovered colon tissue damage, increased level of excitatory neurotransmitters (motilin, gastrin, and substance P) and reduced level of inhibitory neurotransmitters (vasoactive intestinal peptide, somatostatin and endothelin-1) of the mice. Compared with the mice in the Lop group, oral administration of the fermented milk significantly increased the concentrations of acetic acid, propionic acid, butyric acid, isovaleric acid and valeric acid in the feces of the mice, and furthermore, exerted a regulatory effect on the gut microbiota of the mice by up-regulating the abundance of Lactobacillus and Bacteroides, and decreasing the abundance of Helicobacter, Pseudomonas and Porphyromonas. Our results indicated that the combined starter culture fermented milk can effectively alleviate Lop-induced-constipation in BALB/c mice. The relationship between the nutrient profiles and the health promoting function of the yogurt should be further illustrated.