Non-alcoholic fatty liver disease (NAFLD) is becoming an epidemic disease in adults and children worldwide. Importantly, there are currently no approved treatments available for NAFLD. This study aims to investigate the potential applications of sodium tanshinone IIA sulfonate (STS) on improving the NAFLD condition using both in vitro and in vivo approaches. The results showed that STS markedly inhibited lipid accumulation in oleic acid (OA) and palmitic acid (PA) treated HepG2 and primary immortalized human hepatic (PIH) cells. STS suppressed lipogenesis by inhibiting expression of sterol regulatory element binding transcription factor 1 (SREBF1), fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD). In addition, STS reduced inflammation in cells treated with OA-PA, shown by decreased transcriptional levels of tumor necrosis factor (TNF), transforming growth factor beta 1 (TGFB1) and interleukin 1 beta (IL1B). Consistently, protective effects on hepatic steatosis in db/db mice were observed after STS administration, demonstrated by decreased lipid accumulation in mouse hepatocytes. This protective effect might be associated with STS induced activation of sirtuin 1 (SIRT1)/protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1) pathways. Our findings suggest a potential therapeutic role for STS in the treatment of NAFLD.