Tumor-associated macrophages (TAMs) form functionally diverse populations of innate immune cells in the tumor microenvironment. While the pro- and anti-inflammatory TAMs are recognized to shape inflammation and impact disease progression in cancer, the molecular and cellular perturbations of the glycocalyx accompanying TAM polarization remain unknown. Taking a systems glycobiology approach, we here firstly used cell surface-focused glycomics and lectin flow cytometry of ex vivo polarized monocyte-derived macrophages to demonstrate profound sialyl linkage switching of the surface N-glycome in pro-inflammatory (α2,3-NeuAc-favored) and anti-inflammatory (α2,6-NeuAc-dominant) macrophages. No polarization-induced alterations in sialylation were observed in the surface O-glycome. Expression of ST6GAL1 that adds α2,6-sialylation to N-glycans was elevated in anti-inflammatory compared to levels in pro-inflammatory macrophages providing a mechanistic basis for the sialyl linkage switching which was supported by ST6GAL1 silencing. Interestingly, SNA-focused lectin cytochemistry of anti-inflammatory macrophages revealed dense networks of α2,6-sialylated nanotubules forming proteinaceous inter-connecting cellular structures that were absent in pro-inflammatory macrophages. Temporal ST6GAL1 silencing caused nanotubule fragmentation in the anti-inflammatory macrophages. Moreover, live cell recordings of anti-inflammatory macrophages cultured alone and with colorectal cancer (CRC) cells showed reduced macrophage motility and attenuated inter-macrophage and macrophage-CRC cell interactions upon ST6GAL1 disruption indicating functional roles of the α2,6-sialylated nanotubules. Finally, sialyl linkage switching was observed in pro- and anti-inflammatory TAMs using lectin histochemistry on tumor tissues from CRC patients with stage 4 disease. We report on the mechanistic basis for and functional consequences of the profound glycocalyx remodeling accompanying TAM polarization.