Tiller number represents a critical agronomic trait determining sugarcane yield. Strigolactones (SLs) are plant hormones regulating plant architecture. D27, an essential enzyme in the SL biosynthetic pathway, catalyzes a reversible isomerization reaction. ScD27.2, the D27 homolog in sugarcane, contains abiotic stress-responsive elements in its promoter, suggesting its potential importance in SL biosynthesis and stress tolerance. ScD27.2 potentially optimizes sugarcane agronomic traits, particularly tiller number and yield. Understanding its mechanisms will advance the development of high-yielding, stress-tolerant sugarcane varieties. To investigate the role of D27 in sugarcane tillering, the key carotene isomerase gene ScD27.2 was silenced (via RNAi) and overexpressed (OE) in sugarcane cultivar ‘XTT22’ plantlets. ScD27-RNAi-2 sugarcane exhibited decreased ScD27.2 expression and increased tiller numbers compared to the wild type ‘XTT22’. Conversely, overexpression lines (ScD27-OE-1, ScD27-OE-5, and ScD27-OE-9) showed increased ScD27.2 expression and decreased tiller numbers. ScD27-OE-9 demonstrated notable lateral bud germination, while ScD27-RNAi-2 exhibited reduced drought tolerance. Under normal light and water management conditions, transgenic sugarcane plants showed significant differences in tiller number and plant height. During extended drought conditions, ScD27-RNAi-2 height was significantly reduced compared to wild type ‘XTT22’ and ScD27-OE-9, manifesting a dwarf, multi-tiller phenotype. Additionally, ScD27-RNAi-2 showed significantly reduced SLs content. These findings demonstrate that ScD27.2 regulates tillering under drought stress, likely through SL biosynthesis, and that its drought response may be mediated by the transcription factor ScMYB44.