As the neuronal site where voltage gated channel density is highest, the axon initial segment (AIS) plays a key role in establishing a neuron’s action potential threshold, i.e. excitability. Among the properties of AIS that gain attention are length (AISl) and distance from the soma (AISd), which are variously found to change together with neuronal excitability following experimentally-induced perturbations in neural activity. The present study was designed to test the possibility that variation in AIS structural parameters regulates the native range in intrinsic excitability for one class of mature neurons. Spinal motoneurons were selected for their naturally large range in excitability and for their experimental accessibility to in vivo study. We began by determining whether AIS length or distance differed for motoneurons in motor pools that exhibit different activity profiles. Motoneurons sampled from the medial gastrocnemius (MG) motor pool exhibited values for average AISd that were significantly more than for motoneurons from the soleus (SOL) motor pool, which is more readily activated in low-level movements. Next, we tested whether AISd covaried with intrinsic excitability of individual motoneurons. Using anesthetized rats, we measured rheobase current intracellularly from MG motoneurons before labeling them for later immunohistochemical study of AIS. This combinatory approach revealed a significant correlation between AISd and rheobase, for 16 motoneurons sampled within the MG motor pool. Among multiple electrophysiological and morphological parameters measured here, AISd stood out as the dominant predictor of motoneuron excitability. These findings suggest an important role for AISd in setting the intrinsic excitability of spinal motoneurons.