Abstract: Objective To establish a culture system for mouse intestinal organoids and investigate the effect of deoxycholic acid (DCA) on organoids growth. Methods The terminal ileum was collected from 8-month-old C57BL/6 mice. The tissue blocks were treated with EDTA and the crypts were collected and embedded in Matrigel Matrix. Orgnoids growth and buddings were observed in the control group, anhydrous alcohol group, short-term (2 days) 100 μmol/L DCA treatment group, and long-term (10 days) 10 μmol/L DCA treatment group; the orgnoids were further cultured for 10 days after removal of DCA from the medium and observed for orgnoids growth and buddings. Results Short-term treatment with high-concentration DCA resulted in significantly reduced enterosphere formation, enteroids formation, progression from enterospheres to enteroids and number of crypt buds per enteroid (P<0.05), which remained unchanged even after removal of DCA for a short time (P<0.05); long after DCA removal, the enteroids formation rate and number of the crypt buds still remained lower than those in normal organoids (P<0.05). Short-term treatment with low-concentration DCA only resulted in reduced enteroids formation rate and number of crypt buds (P<0.05), and prolonged treatment caused reduced enterospheres formation rate, enteroids formation rate and number of crypt buds (P<0.05). After DCA removal, enteroids formation rate and the number of crypt buds still remained lower than those in the normal group (P<0.05). Conclusion We successfully established an organoids culture system. The presence of DCA in the culture system affects the growth of the organoids, which can partly recover following a prolonged period after the removal of DCA.