Recent research has implicated endocytic pathways as important regulators of receptor signaling. However, the role of endocytosis in regulating chemokine CXC receptor 4 (CXCR4) signaling remains largely unknown. In the present work we systematically investigate the impact of clathrin knockdown on CXCR4 internalization, signaling, and receptor post-translational modification. Inhibition of clathrin-mediated endocytosis (CME) significantly reduced CXCR4 internalization. In contrast to other receptors, clathrin knockdown increased CXCL12-dependent ERK1/2 signaling. Simultaneous inhibition of CME and lipid raft disruption abrogated this increase in ERK1/2 phosphorylation suggesting that endocytic pathway compensation can influence signaling outcomes. Interestingly, using an antibody sensitive to CXCR4 post-translational modification, we also found that our ability to detect CXCR4 was drastically reduced upon clathrin knockdown. We hypothesize that this effect was due to differences in receptor post-translational modification as total CXCR4 protein and mRNA levels were unchanged. Lastly, we show that clathrin knockdown reduced CXCL12-dependent cell migration irrespective of an observed increase in ERK1/2 phosphorylation. Altogether, this work supports a complex model by which modulation of endocytosis affects not only receptor signaling and internalization but also receptor post-translational modification.