The Hedgehog (Hh) signalling pathway plays important roles during embryonic development and in adult tissue homeostasis, for example cartilage, where its deregulation can lead to osteoarthritis (OA). microRNAs (miRNAs) are important regulators of gene expression, and have been implicated in the regulation of signalling pathways, including Hh, thereby impacting upon development and disease. Our aim was to identify the function of miRNAs whose expression is altered in OA cartilage. Here we identified an increase in miR-324-5p expression in OA cartilage and hypothesised that, as in glioma, miR-324-5p would regulate Hh signalling. We determined that miR-324-5p regulates osteogenesis in human mesenchymal stem cells (MSCs) and in mouse C3H10T1/2 cells. Luciferase reporter assays demonstrated that miR-324-5p directly regulated established targets GLI1 and SMO in human but not in mouse, suggesting species-dependent mechanism of Hh pathway regulation. Stable Isotope Labelling with Amino acids in Cell culture (SILAC), mass spectrometry and whole genome transcriptome analysis identified Glypican 1 (Gpc1) as a novel miR-324-5p target in mouse, which was confirmed by real-time RT-PCR, immunoblotting and 3'UTR-luciferase reporters. Knockdown of Gpc1 reduced Hh pathway activity, and phenocopied the effect of miR-324-5p on osteogenesis, indicating that miR-324-5p regulates Hh signalling in mouse via direct targeting of Gpc1. Finally, we showed that human GPC1 is not a direct target of miR-324-5p. Importantly, as well as identifying novel regulation of Indian Hedgehog (Ihh) signalling, this study demonstrates how a miRNA can show conserved pathway regulation in two species but by distinct mechanisms and highlights important differences between human diseases and mouse models.