Synchrotron Microbeam Radiation Therapy (MRT) is an innovative technique that spatially segments the synchrotron radiation field for cancer treatment. A microbeam peak dose is often hundreds of times the dose in the valley (the sub-millimeter region between the peaks of the microbeams). Peak and valley doses vary with increasing depth in tissue which effects tumor dose coverage. It remains to be seen whether the peak or valley is the primary factor in MRT cancer control. This study investigates how unilateral MRT doses can be modulated using a bolus, and identifies the valley dose as a primary factor in MRT cancer control. Fischer rats bearing 9 L gliosarcoma tumors were irradiated with MRT at the Imaging and Medical Beam Line of the Australian Synchrotron. MRT valley doses of 8–15 Gy (250–1040 Gy peak doses) were used to treat tumors with and without a 5 mm dose-modulating bolus. Long-term survival depended on the valley dose primarily (92% correlation), and the use of the bolus reduced the variance in animal survival and improved to the mean survival of rats treated with MRT by 47% and 18% using 15 Gy and 8 Gy valley doses, respectively.