Heat stress (HS) is a critical challenge in broilers due to the high metabolic rate and lack of sweat glands. Results from this study show that implementing a cyclic chronic HS (34 °C for 7 h/d) to finisher broilers decreased the diversity of cecal microbiota and impaired intestinal barrier, resulting in gut leak and decreased body weight (both P < 0.05). These alterations might be related to inflammatory outbursts and the retarded proliferation of intestinal epithelial cells (IECs) according to the transcriptome analysis. Considering the potential beneficial properties of Lactobacillus on intestinal development and function, the protective effects of Lactobacillus rhamnosus (L. rhamnosus) on the intestine were investigated under HS conditions in this study. Orally supplemented L. rhamnosus improved the composition of cecal microbiota and upregulated the transcription of tight junction proteins in both duodenum and jejunum, with a consequent suppression in intestinal gene expressions of pro-inflammatory cytokines and facilitation in digestive capability. Meanwhile, the jejunal villus height of the birds that received L. rhamnosus was significantly higher compared with those treated with the broth (P < 0.05). The expression abundances of genes related to IECs proliferation and differentiation were increased by L. rhamnosus, along with upregulated mRNA levels of Wnt3a and β-catenin in jejunum. In addition, L. rhamnosus attenuated enterocyte apoptosis as indicated by decreased caspase-3 and caspase-9 gene expressions. The results indicated that oral administration with L. rhamnosus mitigated HS-induced dysfunction by promoting intestinal development and epithelial maturation in broilers and that the effects of L. rhamnosus might be dependent of Wnt/β-catenin signaling.