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The incidence of inflammatory bowel diseases (IBD) is increasing worldwide. Mesenchymal stem/stromal cells (MSCs) have immunomodulatory functions and are a promising source for cell transplantation therapy for IBD. However, owing to their heterogeneous nature, their therapeutic efficacy in colitis is controversial and depends on the delivery route and form of transplanted cells. Cluster of differentiation (CD) 73 is widely expressed in MSCs and used to obtain a homogeneous MSC population. Herein, we determined the optimal method for MSC transplantation using CD73+ cells in a colitis model. mRNA sequencing analysis showed that CD73+ cells exhibited a downregulation of inflammatory gene expression and an upregulation of extracellular matrix-related gene expression. Furthermore, three-dimensional CD73+ cell spheroids showed enhanced engraftment at the injured site through the enteral route, facilitated extracellular matrix remodeling, and downregulated inflammatory gene expression in fibroblasts, leading to the attenuation of colonic atrophy. Therefore, the interaction between intestinal fibroblasts and exogenous MSCs via tissue remodeling is one mechanism that can be exploited for colitis prevention. Our results highlight that the transplantation of homogeneous cell populations with well-characterized properties is beneficial for IBD treatment.