Articular chondrocytes experience a transient increase in proliferation and subsequent loss of chondrogenic phenotype in the early stage of osteoarthritis (OA). Pentosan polysulfate sodium (PPS) is a highly sulfated polysaccharide semi-synthetic from glucuronoxylan hemicelluloses, which has been applied for OA management in animals. To investigate the efficacy of different sulfate levels (5, 16%, and 19% as full sulfate level; w/w% of sulfur atoms) of PPS and unsulfured glucuronoxylan (as 0%) on cell cycle regulation and promotion of chondrogenic phenotype in canine articular chondrocytes. Canine chondrocytes were cultured for 24 hr, then incubated with PPS for 72 hr. Chondrocyte viability was measured by a 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cytotoxicity of PPS and cell cycle progression of chondrocytes were checked by flow cytometry. The mRNA levels of cell cycle regulators and chondrocyte phenotypic markers was quantitatively evaluated. Activity of PI3K/Akt pathway was detected by Western blotting. All sulfate levels PPS showed no cytotoxic effect in chondrocytes, while fully sulfated PPS suppressed chondrocyte cell cycle transition from the G1 to later phases at a high concentration. Chondrocytes cultured with full sulfate level PPS significantly reduced cyclin dependent kinase 4 (CDK4) and cyclin D1 (CCND1) mRNA levels, and upregulated type II collagen (COL2A1) and SRY-box 9 (SOX9) expression levels. Akt phosphorylation and PI3K catalytic subunit alpha (p110α) protein production were inhibited in “full” group. PPS suppressed the proliferation in canine articular chondrocytes by targeting PI3K/Akt pathway, and promoted chondrocyte specific gene expression. Full sulfate level maybe necessary for PPS to achieve these effects in chondrocytes.