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The regulatory mechanism of <i>PCSK7</i> gene is still unknown, although its encoded protein PC7 is the most ancient and highly conserved of all proprotein convertases and exhibits enzymatic and non-enzymatic functions in liver triglyceride regulation. Bioinformatics algorithms were used to predict regulatory microRNAs (miRNAs) of <i>PCSK7</i> expression. This led to the identification of four miRNAs, namely miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p, with potential binding sites on the 3′-untranslated region (3′-UTR) of human <i>PCSK7</i> mRNA. The expression patterns of these miRNAs and <i>PCSK7</i> mRNA were assessed in three different cell lines with quantitative polymerase chain reaction (qPCR), which revealed reciprocal expression patterns between the expression levels of the four selected miRNAs and <i>PCSK7</i>. Next, the interactions and effects of these miRNAs on <i>PCSK7</i> expression levels were investigated via cell-based expression analysis, dual-luciferase assay, and Western blot analysis. The data revealed that <i>PCSK7</i> mRNA levels decreased in cells transfected with vectors overexpressing miR-125a-5p, miR-143-3p, and miR-409-3p, but not miR-320a-3p. The dual-luciferase assay demonstrated that the above three miRNAs could directly interact with putative target sites in <i>PCSK7</i> 3′-UTR and regulate its expression, whereas miR-320-3p exhibited no interaction. Western blot analysis further revealed that the overexpression of miR-125a-5p in Huh7 cells inhibits the expression and ability of PC7 to cleave human transferrin receptor 1. Our results support a regulatory role of these miRNAs on <i>PCSK7</i> expression and function and open the way to assess their roles in the regulation of PC7 activity in vivo in the development of hepatic steatosis.