<p>Chromatin accessibility is a key determinant of transcriptional regulation across eukaryotes, yet its organization and functional significance in ciliated protists remain poorly defined. Here, we optimized ATAC-seq to delineate the chromatin landscape in the transcriptionally active macronucleus of <i>Tetrahymena thermophila</i>. Our analyses reveal that accessible chromatin is predominantly enriched upstream of transcription start sites (TSSs), in stark contrast to the promoter-proximal accessibility observed in human embryonic stem cells. Dynamic profiling across different life cycle stages of <i>T. thermophila</i> (vegetative growth, starvation, conjugation, and refeeding) uncovers coordinated shifts in chromatin architecture that closely mirror extensive transcriptional reprogramming. Moreover, transcriptional inhibition by flavopiridol reduces chromatin openness upstream of TSSs while enhancing nucleosome organization downstream of TSSs, underscoring the direct impact of transcription on shaping chromatin structure. Comparative analyses across 13 eukaryotic species further indicate that upstream-biased accessibility is an ancestral trait in unicellular eukaryotes, whereas higher vertebrates have evolved more complex promoter architectures. These findings not only establish <i>T. thermophila</i> as a powerful model for dissecting chromatin-dependent gene regulation but also provide novel insights into the evolutionary divergence of chromatin organization across the eukaryotic lineage.</p>