Unpasteurized milk consumption was associated with less atopy prevalence. Not only microbial load but also fatty acids and cytokines such as transforming growth factor-β1 (TGF-β1) may play a role on the effect of unpasteurized milk. Levels of TGF-β1 in different cow’s milk samples were evaluated: we consider raw unpasteurized milk before and after boiling, commercial pasteurized and micro-filtrated cow’s milk and different commercially available cow’s milk formulas. TGF-β1 concentration in raw unpasteurized cow’s milk was 642.0 ± 52.9 pg/ml before boiling and decreased significantly after boiling (302.7 ± 50.59 pg/ml) (p < 0.05). TGF-β1 concentrations were also significantly lower in commercial pasteurized milk (246.2 ± 43.15 pg/ml) and in commercial micro-filtrated milk (213.0 ± 31.6 pg/ml) in comparison to unpasteurized unboiled milk (p = 0.002). The levels of TGF-β1 in all formula samples were below the threshold of detectability for the assays. As TGF-β1 in the milk may contribute to the development of the immature gastrointestinal tract by influencing IgA production and oral tolerance induction, we suggest to consider not only the microbial compounds but also the cytokine patterns to explain the protective effect of unpasteurized cow’s milk on allergic disorders.