Renal interstitial fibrosis (RIF) is a common feature that facilitates the progression of chronic kidney disease (CKD), and emerging lines of evidence suggest that microRNA-376b (miR-376b) is capable of promoting RIF. In this study, we examined collagen deposition in kidney tissues, the autophagy and mitochondrial reactive oxygen species (ROS) of macrophages, and the apoptosis of kidney fibroblasts (KFBs) after the promotion or suppression of endogenous miR-376b in cultured macrophages and renal fibroblasts obtained from mice with CKD. FVB/N mice were prepared to establish a CKD model. A target prediction program and luciferase activity determination were used to confirm that autophagy-related gene 5 (Atg5) was a direct target of miR-376b. Macrophages and KFBs were isolated after the treatment to study the mechanisms and functions of miR-376b in relation to Atg5 in CKD. The autophagy level was determined, and KFB proliferation and apoptosis were assessed through MTT and EdU assays and flow cytometry, respectively. Atg5 was confirmed as a direct target of miR-376b. miR-376b and Atg5 exhibited high and low expression in kidney tissues from mice with CKD. The mice treated with a miR-376b inhibitor exhibited reduced collagen deposition, suppressed interstitial fibrosis, a higher level of autophagy, higher ROS production, enhanced apoptosis, and inhibited proliferation of KFBs, which suggested that the downregulation of miR-376b could exert beneficial effects on CKD through Atg5. miR-376b downregulation promotes macrophage autophagy to relieve RIF by negatively regulating Atg5 in mice with CKD. Thus, miR-376b might represent a potential focus of future investigations on treatments for CKD.