Background: Rapamycin is produced from Streptomyces hygroscopicus, and was initially identified as an antifungal antibiotic. More recently, rapamycin has been found to have various medical applications, including in relation to immunosuppression and anti-aging. Due to its complex structure, biological production is the major route for commercialized rapamycin production. The conventional fermentation process requires a large seed fermenter for the inoculation process (in general, the volume of the seed fermenter is equal to 5-10 % that of the production fermenter), which presents challenges with regard to scaling up production, due to the high investment costs of seed fermenters. This study explored different inoculation strategies for rapamycin production in a 15-L agitation fermenter. Results:The results indicated that solid-state fermentation (SSF) using barley as the substrate is a suitable method for the inoculation. The highest rapamycin concentration measured in the batch with SSF (barley) inoculated was about 520 mg/L, which was significantly higher than that of 400 mg/L obtained in the batch inoculated with 5 % liquid seed medium. Besides the higher rapamycin production, using SSF of barley as the inoculation method can greatly reduce both the labor and cost requirements. Conclusions:The usage of mycelium-covered barley as the solid substrate for the inoculation of 15-L fermenter leading to a higher rapamycin production compared to that of conventional liquid seed medium. The solid-state inoculation method can avoid both the intensive labor requirement and costly seed fermenter needed with the latter approach. This inoculation method thus has the potential to be applied to the large-scale production of rapamycin.