High-fat diet (HFD) induces imbalance in the small intestine environment, where fat digestion and absorption mainly take place. This study aimed to elucidate the mechanisms by which Lonicera caerulea polyphenols (LCP) might inhibit fat absorption, from the perspective of small intestine microbiota and epithelial barrier integrity. Male Sprague-Dawley rats were given HFD with or without co-administration of LCP for 8 weeks. The results showed that LCP supplementation significantly decreased the levels of serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C), and increased the contents of fecal sterols, in HFD rats. LCP also inhibited the dysfunction of the small intestine epithelial barrier, via alleviating the oxidative stress activated by Nrf2-ARE pathway, and by modulating the expressions of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), nuclear factor kappa-B p65 (NF-κB p65) and inducible nitric oxide synthase (iNOS) in the small intestine. Additionally, LCP administration restored the balance in small intestine microbiota and increased the abundance of the specific bacteria, such as Lactobacillus, involved in fat absorption. Our results demonstrated that LCP may be beneficial to inhibit fat absorption. The mechanism seems to be associated with the protection of the epithelial barrier integrity and the modulation of specific bacteria in the small intestine.