In order to evaluate the effect of non-viable Lactobacillus paracasei L9 (L9) in normal temperature yogurt on relaxing constipation and modulating gut microbiota, sixty-one subjects with constipation were recruited and divided into two groups, drinking normal temperature yogurt or yogurt containing non-viable L9 for 28 days, respectively. Constipation-related symptoms, the content of short chain fat acids (SCFAs) and variation of gut microbiota were analyzed. Compared to the group drinking normal temperature yogurt, the results in group drinking yogurt with non-viable L9 were as follows:the defecation frequency was significantly increased (P<0.05), the defecation condition and consistency scores were remarkably reduced (P<0.01), the content of butyric acid was dramatically increased (P<0.01), propionic acid and total acid were significantly increased (P<0.05), and bacterial species producing SCFAs such as Blautia, Ruminococcus_2 and _hallii_group were significantly increased. The results indicated that normal temperature yogurt with non-viable L9 could help to relieve constipation and modulate gut microbiota, which might provide a theoretical basis for developing functional foods containing non-viable L9.