Ergothioneine is a natural compound with strong antioxidant properties. In this study, the effects of different nitrogen sources including ammonium nitrate, ammonium sulfate, sodium nitrate, and histidine in sawdust media were investigated to enhance ergothioneine contents in Shiitake (Lentinula edodes) fruit bodies. The addition of 0.2% ammonium sulfate in the growth media showed the highest enhancement of ergothioneine content in shiitake fruit bodies which was 1.7-fold higher than the control. On the other hand, histidine, a building block of ergothioneine decreased the concentration of ergothioneine significantly. Our results demonstrate that the cultivation of shiitake in sawdust media with suitable nutrients was effective to enhance its ergothioneine contents.