<p>Brassinosteroid (BR) exert a crucial regulatory role in shaping plant architecture. However, the BR biosynthesis and underlying genetic variations governing potato plant architecture remain poorly understood. Here, we identified a single recessive locus, <i>ap</i>, which controls the abnormal architectural plant (AP) with dark-green curled leaves, shortened internodes, and delayed flowering, using a BC₄S₁ population. The functional gene, <i>StCYP90C1</i>, encoding 3-epi-6-deoxocathasterone 23-monooxygenase, was identified at this locus. <i>StCYP90C1</i> expression was significantly lower in&#xa0;the leaves of APs than in normal architectural plants (NPs), and this reduced expression was identified to be associated with two key variations in the promoter region of APs. CRISPR/Cas9-mediated knockout of <i>StCYP90C1</i> resulted in typical BR-deficient traits, including dark-green curled leaves, shortened internodes, and dwarfism. Quantification of BR intermediates revealed that the levels of 6-deoxocathasterone (6-dexoCS) and cathasterone (CS) were significantly lower in <i>stcyp90c1</i>-ko mutants compared to the wild type. Transcriptome analysis revealed some candidate genes associated with plant morphogenesis in the knockout mutant. This study elucidates the important role of <i>StCYP90C1</i> in modulating plant architecture via BR biosynthesis and identifies a deleterious mutation locus that should be excluded from inbred line development in potato breeding.</p>