Major advance was done in preclinical radiotherapy thanks to the development of image guided micro-irradiator. Nevertheless, some applications still can benefit of improvements, such as the irradiation of mobile tumors. This preclinical radiotherapy case presents increased difficulties compared to clinical practice because of the waveform of small animals breathing cycle, its frequency and amplitude. To answer this issue, we developed a specific beam shutter and implemented respiratory gating on the X-RAD 225Cx preclinical irradiator. In the first step of this study, the shutter was accurately characterized. Opening and closing speed of 1.28 and 0.33 mm ms−1 were respectively measured, and a transmission of 0.7% of the beam was measured with the shutter fully closed. Beam-on times were also determined for various gating parameters and highlighted a difference of 57 ms between the beam delivery duration and the gate width. This discrepancy was compensated during the respiratory monitoring adjustment. In a second step, a respiratory protocol was evaluated with two vertical beams of 2.5 and 5 mm diameters, for motion amplitudes ranging from 0.5 to 4 mm. This evaluation demonstrated the effectiveness of our set up to perform motion compensation for amplitude as small as 0.5 mm despite a dose gradient of 1.47 cGy mm−1 observed with the 5 mm irradiation field, due to the shutter opening and closing durations. We also investigated the efficiency of a scintillating fiber dosimeter, adapted to small beams and providing real-time dose rate measurements. This detector showed very good performances to detect motion in small irradiation fields and would be very suitable to monitor the number of delivered gates until the planned delivered dose is achieved. This study presented a new respiratory gating set up and showed that very efficient motion compensation could be achieved in small animal radiotherapy.