MicroRNAs (miRNAs) are promising biomarkers for different pathological models because of their stable and detectable characters in biofluids. Here, we collected urine samples from 5 beagle dogs on the 3th, 6th, and 12th day in an acute kidney injury (AKI) caused by gentamycin. miRNA levels were measured with high-throughput sequencing and the results were then differentially investigated. Gene Ontology (GO) and KEGG pathway analysis were performed to analyze potential target genes corresponding to the differentially expressed miRNAs (DE-miRNAs). Relationships between hub genes and DE-miRNAs were analyzed with STRING and Cytoscape. We identified 234 DE-miRNAs 3, 6, and 12 days after gentamycin treatment (p < 0.05). Top 10 up- and down-regulated candidate target genes of DE-miRNAs were predicted by overlapping TargetScan and miRanda results). GO and KEGG analyses for DE-miRNAs demonstrated that the DE-miRNAs target genes are mainly involved in kidney injury-related pathways, such as the insulin signaling pathway, oxytocin signaling pathway, and hedgehog signaling pathway. The network of miRNA-hub genes suggests that miR-452, miR-106a, and 106b participate in regulating the largest number of hub genes. We evaluated the miRNA signature via a canine model built by gentamycin-caused acute kidney injury. Our results represent a valuable resource for evaluating miRNAs as biomarkers of renal toxicity.