Background: miR-181a is a small non-coding RNA known to be dysregulated in osteoarthritis (OA), but the role of miR-181a in human OA remains unclear. The aim of this study was to identify its function and molecular target in chondrocytes during OA pathogenesis. Material/Methods: The function of miR-181a was assessed by gain-of-function studies in human OA chondrocytes. Potential targets of miR-181a were predicted using series of bioinformatics and intersection analysis, then confirmed by luciferase reporter assay. Gene expression was quantified using quantitative reverse transcription PCR (qRT-PCR) assays, and protein production was quantified by Western blot analysis. Results: The FITC apoptosis assay results indicated that the upregulation of miR-181a led to an increase of apoptosis rate in chondrocytes. Then bioinformatic analysis identified potential target sites of the miR-181a located in the 3' untranslated region of GPD1L. Dual-luciferase reporter assays results showed that GPD1L is a target gene of miR-181a. Furthermore, Western blot and qRT-PCR analysis demonstrated that miR-181a inhibited GPD1L gene expression. Increased GPD1L and decreased miRNA-181a were observed in tissues from osteoarthritis patients. Moreover, we found a highly negative correlation between miRNA-181a and GPD1L. Conclusions: Our results demonstrated that miR-181a may play an important role in the pathogenesis of OA through targeting GPD1L and regulating chondrocyte apoptosis.