MiRNA is a class of small non-coding RNA which has an important effect on posttranscriptional gene regulation. It can regulate the expression of the target gene at the mRNA level and further influence the protein level of the target gene. We found that ULK1 may be the target gene of miR-26a-5p, and ULK1 (unc-51 like autophagy activating kinase 1) is a key component in autophagy pathway. In this study, we overexpressed miR-26a-5p by transfecting miR-26a-5p mimic into cells and simultaneously inhibited miR-26a-5p by transfecting miR-26a-5p inhibitor into cells. We demonstrated that overexpression of miR-26a-5p can reduce the expression of ULK1 and collagen I, and decrease the activation of LC3-I to LC3-II. In contrast, inhibition of miR-26a-5p can increase the expression of ULK1 and collagen I, and increase the activation of LC3-I to LC3-II. The Dual-luciferase reporter assay showed that miR-26a-5p directly acted on the 3′UTR of ULK1 and thus affected the expression of ULK1. As such, our study demonstrated that miR-26a-5p might regulate the autophagy in cardiac fibroblasts by targeting ULK1, which may have an effect on cardiac fibrosis. To our knowledge, this is the first study that shows miR-26a-5p regulates the autophagic pathway in cardiac fibroblasts.