BACKGROUND A significant trend towards increased fusidic acid (FA) resistance among Staphylococcus aureus with increased duration of use is of concern. The aim of the present study is to investigate the dissemination of fusidic acid resistance among Staphylococcus aureus clinical isolates. METHODS The susceptibility of S. aureus isolates to antimicrobial agents was determined by disc-diffusion method. The minimal inhibitory concertrations(MICs) of fusidic acid and vacomycin for fusidic acid-resisitant isolates were determined by ager dillution method. FA resistance determinants were determined by PCR and DNA sequencing. SCCmec typing, spa typing and multi-locus sequence typing were used for the determination of molecular characteristics for S. aureus isolates. RESULTS A total of 392 non-duplicate S. aureus isolates including 181 methicillin-resistant S. aureus (MRSA) isolates, which were isolated from the clinical specimens of patients at a Chinese tertiary hospital from January, 2012 to September, 2013, were collected for investigating FA resistance. Among 392 S. aureus clinical isolates tested, 56 (14.3%) with FA MIC values ranging from 2 μg/ml to ≥128 μg/ml were resistant to FA. The proportions of FA resistance among MRSA and MSSA isolates were 27.1% (49/181) and 3.3% (7/211). There was a trend of rapidly increased FA resistance among S. aureus and MRSA isolates from 5.2% and 8.9% in 2012 to 24.9% and 45.1% in 2013. Acquired FA resistance gene, fusB, was present in 73.2% (41/56) of FA-resistant S. aureus isolates. fusC and fusA mutation were not found in any of tested isolates. A total of 9 sequence types (STs) and 12 spa types were identified among the 56 FA-resistant S. aureus isolates. ST5 accounting for 66.1% (37/56) was the most prevalent ST. The majority (92.9%, 52/56) of the isolates tested belonged to clonal complex 5(CC5). t2460 was the most prevalent spa type, accounting for 67.9% (38/56) . ST5-MRSA- II-t2460 was predominant clone, accounting for 75.5% (37/49) of FA-resistant MRSA isolates and 66.1% (37/56) of FA-resistant S. aureus isolates. Five of 7 FA-resistant MSSA isolates belonged to ST630-MSSA. CONCLUSIONS Increased FA resistance among S. aureus isolates was found in China. fusB was predominant FA resistance determinant. The spread of CC5 clone, especially novel ST5-MRSA- II-t2460 clone with high-level resistance to FA, was responsible for the increase of FA resistance.