Toxocara canis is of significant public health concern and toxocariasis, one of the most neglected zoonoses in the world, has gained an increasing international attention. To date, neither miRNA expression profiling in spleen has been reported in dogs after T. canis infection to date. In this study, we performed sequencing-based transcriptome profiling of miRNA expression in the spleen of Beagle puppies after T. canis infection at 24 hours post-infection (hpi), 96 hpi, and 36 days post infection (dpi), in order to get a better understanding of the related miRNAs and their regulatory network of target genes. And 20, 34 and 19 differentially expressed (DE) miRNAs at the three stages of 24 hpi, 96 hpi and 36 dpi, respectively, and interactions with their potential targets were identified. The present study revealed that marked alterations in the expression profiles of spleen miRNA, with possible implications in the pathophysiology of toxocariasis.