Milk-derived extracellular vesicles (MEVs) combat acute and chronic pro-inflammation in peripheral cells and tissues. However, the biological functions of MEVs in the central nervous system require exploration. We investigated whether MEVs activate the heat shock response (HSR) in polarized human microglia. MEVs were isolated from unpasteurized human donor milk (n=12 anonymous donors). Human microglia clone 3 cells were primed with 10 ng/mL interferon-gamma to induce polarization, and a subset of cells was supplemented with 200 µg of MEVs. The abundance of HSF1 and candidate heat shock proteins (Hsp70, Hsp90, Hsp40, Hsp27) was analyzed using quantitative reverse transcription polymerase chain reaction and western immunoblotting at 6 h, 12 h, and 24 h post-MEV treatment. We found that MEV treatment promoted the HSR in polarized microglia, compared to homeostatic cells. Furthermore, MEVs increased the duration of the HSR in polarized microglia, exerting robust and continued pro-survival benefits.