This study investigated the effects of diet supplementation with alkaline protease (AKP) on the production performance, egg quality, and cecal microbiota of laying hens. A total of 720 Hy-Line Brown laying hens (60 weeks old) were divided into four groups with six replicates of 30 birds each. No AKP was added to the control diet, and the hens in the other three groups (Groups 1, 2, and 3) were fed the basal diet supplemented with AKP preparations at 3, 6, and 9 u/g of diet, respectively. Results showed that AKP supplementation significantly decreased the feed/egg ratio (p < 0.05). Compared with that of the control group, the eggshell strength of Group 1 was significantly increased (p < 0.05), and the egg yolk weight of Groups 1 and 3 was significantly increased (p < 0.05). Distinctive difference in cecal microbiota was observed between AKP and control groups, and the average values of microbial diversity was lower in the AKP group than in the control group. The relative abundance of Bacteroidetes and Firmicutes at the phylum level, Rikenellaceae, Lachnospiraceae, Lactobacillaceae, Erysipelotrichaceae, and Christensenellaceae at the family level, and Rikenellaceae_RC9_gut_Group, Lactobacillus, Romboutsia, Lachnoclostridium, and Blautia at the genus level in the AKP group changed significantly compared with that in the control group (p<0.05).