Purpose X-ray microbeam radiation therapy is a preclinical concept for tumor treatment promising tissue sparing and enhanced tumor control. With its spatially separated, periodic micrometer-sized pattern, this method requires a high dose rate and a collimated beam typically available at large synchrotron radiation facilities. To treat small animals with microbeams in a laboratory-sized environment, we developed a dedicated irradiation system at the Munich Compact Light Source (MuCLS). Methods A specially made beam collimation optic allows to increase x-ray fluence rate at the position of the target. Monte Carlo simulations and measurements were conducted for accurate microbeam dosimetry. The dose during irradiation is determined by a calibrated flux monitoring system. Moreover, a positioning system including mouse monitoring was built. Results We successfully commissioned thein vivomicrobeam irradiation system for an exemplary xenograft tumor model in the mouse ear. By beam collimation, a dose rate of up to 5.3 Gy/min at 25 keV was achieved. Microbeam irradiations using a tungsten collimator with 50 mu m slit size and 350 mu m center-to-center spacing were performed at a mean dose rate of 0.6 Gy/min showing a high peak-to-valley dose ratio of about 200 in the mouse ear. The maximum circular field size of 3.5 mm in diameter can be enlarged using field patching. Conclusions This study shows that we can performin vivomicrobeam experiments at the MuCLS with a dedicated dosimetry and positioning system to advance this promising radiation therapy method at commercially available compact microbeam sources. Peak doses of up to 100 Gy per treatment seem feasible considering a recent upgrade for higher photon flux. The system can be adapted for tumor treatment in different animal models, for example, in the hind leg.