Constipation is a common disorder that is a significant source of morbidity among people around the world ranging from 2% to 28%. Dendrobium officinale Kimura et Migo is a traditional herbal medicine and health food used for tonicity of the stomach and promotion of body fluid production in China. This study aimed to prepare the ultrafine powder of Dendrobium officinale (UDO) and investigate its laxative effect and potential mechanism in mice with diphenoxylate-induced constipation. Results indicated that the mean diameter (d(50)) of UDO obtained by ball milling was 6.56 mu m. UDO (62.5, 125, and 250 mg/kg, p.o.) could significantly enhance the gastrointestinal transit ratio and promote fecal output. Moreover, UDO treatment resulted in significant increases in the serum levels of acetylcholinesterase (AChE), gastrin (Gas), motilin (MTL), and substance P (SP), and obviously decreased serum contents of somatostatin (SS). Taken together, UDO, which can be easily obtained through milling to a satisfactory particle size, exhibited obvious laxative effect in diphenoxylate-induced constipated mice, and the mechanism might be associated with elevated levels of AChE, Gas, MTL, SP, and reduced production of SS. UDO has the potential for further development into an alternative effective diet therapy for constipation.