Slow-transit constipation (STC) is a debilitating disorder of colonic motility with poorly defined molecular underpinnings. While abnormalities in the enteric nervous system and interstitial cells of Cajal have been extensively studied, smooth-muscle–intrinsic mechanisms remain largely unexplored. Here, we combine spatially resolved proteomics of human colon with functional perturbations in primary human colonic smooth muscle cells (HcoSMCs) to uncover two regulators of motility: Bridging Integrator 1 (BIN1) and Aldehyde Dehydrogenase 1B1 (ALDH1B1). Both proteins were downregulated in colonic smooth muscle of STC patients, localized to fibrotic regions. Lentiviral knockdown of BIN1 or ALDH1B1 in HcoSMCs impaired ATP-evoked Ca²⁺ responses and contraction, disrupted mitochondrial architecture, and elevated reactive oxygen species. Mechanistically, BIN1 loss coincided with mitochondrial apoptotic signaling and extracellular matrix deposition, whereas ALDH1B1 loss induced mitophagy and NF-κB–driven inflammatory programs. Transcriptomic analyses further revealed convergence on profibrotic pathways, consistent with collagen accumulation observed in patient tissues. Together, our findings delineate a smooth-muscle–centric framework of STC pathogenesis and nominate BIN1 and ALDH1B1 as candidate regulators of intestinal motility and potential entry points for therapeutic intervention.