Damage to the lateral hypothalamus (LH) causes profound physical inactivity in mammals. Several molecularly distinct types of LH neurons have been identified, including orexin cells and glutamic acid decarboxylase 65 (GAD65) cells, but their interplay in orchestrating physical activity is not fully understood. Here, using optogenetic circuit analysis and cell type-specific deep-brain recordings in behaving mice, we show that orexin cell activation rapidly recruits GAD65LH neurons. We demonstrate that internally initiated GAD65LH cell bursts precede and accompany spontaneous running bouts, that selective chemogenetic silencing of natural GAD65LH cell activity depresses voluntary locomotion, and that GAD65LH cell overactivation leads to hyperlocomotion. These results thus identify a molecularly distinct, orexin-activated LH submodule that governs physical activity in mice.