Chemotherapy-induced alopecia (CIA) represents the most distressing side-effect for cancer patients. Scalp cooling is currently the only treatment to combat CIA, yet little is known about its cytoprotective effects in human hair follicles (HF). We have previously established in vitro human keratinocyte models to study the effects of taxanes and anthracyclines routinely-used clinically and reported that cooling markedly-reduced or even completely-prevented cytotoxicity in a temperature dependent manner. Using these models (including HF-derived primary keratinocytes), we now demonstrate that cooling markedly attenuates cellular uptake of the anthracyclines doxorubicin and epirubicin to reduce or prevent drug-mediated human keratinocyte cytotoxicity. We show marked reduction in drug uptake and nuclear localization qualitatively by fluorescence microscopy. We have also devised a flow cytometry-based methodology that permitted semi-quantitative analysis of differences in drug uptake, which demonstrated that cooling can reduce drug uptake by up to ~8-fold in comparison to normal/physiological temperature, an effect that was temperature-dependent. Our results provide evidence that attenuation of cellular drug uptake represents at least one of the mechanisms underpinning the ability of cooling to rescue human keratinocytes from chemotherapy drug-cytotoxicity, thus supporting the clinical efficacy of scalp cooling.