Fetal mesenchymal stem/ stromal cells (fMSC) represent a developmentally-advantageous cell type with translational potential. CXCR4, the receptor for the chemokine SDF-1α, is implicated in migration of MSC to bone marrow and sites of tissue injury, but low surface expression impairs targeting strategies to meet tissue repair requirements. We investigated trafficking of CXCR4 in human fetal bone marrow fMSC and its importance in CXCR4 surface expression and function. As in adult MSC, CXCR4 surface expression in fMSC was low (3-4% of cells), with most CXCR4 protein sequestered intracellularly (>90%). Using immunofluorescence microscopy, we showed that CXCR4 has a punctate intracellular distribution pattern similar to Rab5 and Rab11, markers of early and recycling endosomes respectively. The late endosome/lysosomal marker Lamp1 showed a distinct but overlapping intracellular localization pattern to CXCR4. Co-localization of CXCR4 with Rab5, Rab11 and Lamp1 indicated an even distribution between all three endosomal compartments. We next demonstrated that endocytosis of CXCR4 is largely independent of endogenously-produced SDF-1. Treatment of fMSC with two inhibitors of endocytosis, blebbistatin or dynasore increased CXCR4 surface expression at least 5 fold, from 4.8±0.5% to 33.4±11.3% and 25.6%±5.4 for blebbistatin and dynasore respectively, p<0.05. Furthermore both inhibitors enhanced migration of fMSC toward SDF-1α in in vitro chemotaxis assays (≥2.6 fold, p<0.01). Kinetic modeling of recycling and endocytosis rates informed by these data confirmed a state of accelerated internalization of CXCR4 in resting fMSC. These data implicate constitutive endocytosis in the regulation of CXCR4 membrane expression, and suggest pharmacological strategies to enhance migration of systemically-transplanted cells.