We describe a wide-field optical tomography technique, which allows the measurement-guided optimization of illumination patterns for enhanced reconstruction performances. The iterative optimization of the excitation pattern aims at reducing the dynamic range in photons transmitted through biological tissue. It increases the number of measurements collected with high photon counts resulting in a dataset with improved tomographic information. Herein, this imaging technique is applied to time-resolved fluorescence molecular tomography for preclinical studies. First, the merit of this approach is tested by in silico studies in a synthetic small animal model for typical illumination patterns. Second, the applicability of this approach in tomographic imaging is validated in vitro using a small animal phantom with two fluorescent capillaries occluded by a highly absorbing inclusion. The simulation study demonstrates an improvement of signal transmitted (∼2 orders of magnitude) through the central portion of the small animal model for all patterns considered. A corresponding improvement in the signal at the emission wavelength by 1.6 orders of magnitude demonstrates the applicability of this technique for fluorescence molecular tomography. The successful discrimination and localization (∼1 mm error) of the two objects with higher resolution using the optimized patterns compared with nonoptimized illumination establishes the improvement in reconstruction performance when using this technique.