Silk fibroin (SF) thin films were prepared by pulsed IR laser deposition. Size variation of the smallest protein units (SPU) in the films were evaluated by atomic force microscopy. Average SPU size doubled with increasing the background gas pressure by an order of magnitude. The size of SPU was significantly larger when the film was deposited in Ar atmosphere than in He while the pressure was kept constant at 100Pa. We discussed about the growth of SPU in terms of ionization as a consequence of hydrogen bond rupture by laser irradiation, with the aid of infrared spectra. Effects of the background gas were suspected primarily to be the difference in the states of collision with the fragmented protein units immediately after the laser bombardment of the target, within or near the plume zone. The results suggest the possibility of controlling the nano-structures of the protein thin film by optimizing the background gas condition.