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In Mexico, infants (0–2 years old) show the highest prevalence of vitamin A deficiency (VAD), measured by serum retinol concentrations. Thus, we consider that low vitamin A (VA) intake through breast milk (BM) combined with poor weaning practices are the main factors that contribute to VAD in this group. We combined the assessment of VA status in lactating women using BM retinol and a stable isotope ‘dose-to-mother’ technique to measure BM production in women from urban and agricultural areas. Infants’ mean BM intake was 758 ± 185 mL, and no difference was observed between both areas (p = 0.067). Mean BM retinol concentration was 1.09 μmol/L, which was significantly lower for the agricultural area (p = 0.028). Based on BM retinol concentration, 57% of women were VAD; although this prevalence fell to 16% when based on fat content. Regardless of the VA biomarker used here, infants from the urban and agricultural areas cover only 66% and 49% of their dietary adequate intake from BM, respectively (p = 0.054). Our data indicate that VAD is still a public health concern in Mexico. Adopting both methods to assess VA transfer from the mother to the breastfed child offers an innovative approach towards the nutritional assessment of vulnerable groups.