Microtubules have been extensively studied for their interactions at the outer surface and ends, yet the regulatory mechanisms within the microtubule lumen, particularly in singlet microtubules, remain poorly understood. Here, we developed a proximity-labeling strategy to distinguish intraluminal from external proteins, enabling the systematic identification of key regulators residing within the singlet microtubule lumen. Among these, we focused on Jupiter microtubule-associated homolog 2 (JPT2), a conserved microtubule-binding protein. JPT2 binds directly to microtubules and localizes to the lumen in a C-terminal-dependent manner. Within this compartment, it modulates the distribution of the α-tubulin acetyltransferase MEC17 and contributes to luminal homeostasis. Notably, the luminal accessibility of JPT2 is highly sensitive to microtubule structural states and is markedly reduced by Paclitaxel treatment, suggesting that drug-induced conformational changes or steric hindrance can restrict luminal entry. Together, our findings identify JPT2 as a key organizer of the singlet microtubule lumen and provide a framework for the spatial and functional analysis of microtubule inner proteins. Beyond exogenous perturbations, our observations suggest that luminal protein distribution may also be modulated by steric competition or selective entry among intraluminal components, indicating a more intricate regulatory landscape within the microtubule interior.