Objective · To explore the effects of maternal vitamin D deficiency before and during pregnancy on the intestinal flora and cathelicidin antimicrobial peptide (CAMP) in offspring rats. Methods · Twenty-four female Sprague-Dawley rats (8-week-old) were randomly assigned to three groups (n8 per group), i.e. control group (C group), vitamin D deficiency group (VDD group), and vitamin D supplement group (VDS group). Special diets were used to build the rat models of vitamin D deficiency before and during pregnancy. Maternal 25(OH)D level was detectedliquid chromatography tandem mass spectrometry (LC-MS/MS) at gestational day 14.At the age of 4 weeks in offsprings, the level of 25(OH)D was measured; the faeces were collected for the detection of intestinal flora; the mRNA and protein of CAMP were evaluatedreal-time quantitative RTPCR and Western blotting, respectively. Results · The rat models of vitamin D deficiency before and during pregnancy were successfully established.The relative abundance of intestinal Lactobacillus in theoffspring rats in C group,VDD group andVDS group was 0.050±0.016,0.028±0.013 and0.033±0.021, respectively. Compared with C group, the relative abundance of intestinal Lactobacillus in VDD group significantly decreased (P<0.05)，and the mRNA and protein of colonic CAMPsignificantly decreased as well (P<0.05). Compared with VDD group, VDS group showed a trend of increase in the abundance of intestinal Lactobacillus;themRNA ofcolonic CAMPdidn’tchange significantly, but the proteinof colonic CAMP significantly increased (P<0.05). Conclusion · Vitamin D deficiency during pregnancy results in decreased abundance of intestinal Lactobacilli as well as reduced mRNAand protein of colonic CAMPin offspring rats. Vitamin D supplementation significantly improves the protein of colonic CAMP and induces a trend of increase in the abundance of intestinal Lactobacilli in offspring rats.