Stem structural failure, or lodging, affects many crops including sorghum, and can cause large yield losses. Lodging is typically caused by mechanical forces associated with severe weather like high winds, but exposure to sub-catastrophic forces may strengthen stems and improve lodging resistance. The responses of sorghum internodes at different developmental stages were examined at 2 and 26 h after initiating moderate mechanical stimulation with an automated apparatus. Transcriptome profiling revealed that mechanical stimulation altered the expression of over 900 genes, including transcription factors, cell wall-related and hormone signaling-related genes. IAA, GA1 and ABA abundances generally declined following mechanical stimulation, while JA increased. Weighted Gene Co-expression Network Analysis (WGCNA) identified three modules significantly enriched in GO terms associated with cell wall biology, hormone signaling and general stress responses, which were highly correlated with mechanical stimulation and with biomechanical and geometrical traits documented in a separate study. Additionally, mechanical stimulation-triggered responses were dependent on the developmental stage of the internode and the duration of stimulation. This study provides insights into the underlying mechanisms of plant hormone-regulated thigmomorphogenesis in sorghum stems. The critical biological processes and hub genes described here may offer opportunities to improve lodging resistance in sorghum and other crops.