The underlying source of negative BOLD responses (NBRs) is still debated. Here, we show that binocular visual stimulation with short inter-stimulus-intervals (ISIs) lead to strong NBRs in the rat superior colliculus (SC). We perturbed cortical feedback loops with V1 ibotenic acid lesions and input to the ipsilateral SC via tectotectal projections through monocular stimulation. SC NBRs were upward regulated upon both V1 lesioning and monocular stimulation of non-lesioned animals. When combining reduced cortical feedback and tectotectal projections, NBRs in SC were abolished. Our results suggest an important role for corticotectal and commissural tectotectal projections in SC NBRs at short ISIs.