The transfer of the perfluoroalkyl acids (PFAAs) perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS), perfluorooctanesulfonate (PFOS), and perfluorooctanoate (PFOA) from feed into tissue and milk of dairy cows was investigated. Holstein cows (n = 6) were fed a PFAA-contaminated feed for 28 days. After the PFAA-feeding period, three cows were slaughtered while the others were fed PFAA-free feed for another 21 days (depuration period). For PFAA analysis plasma, liver, kidney, and muscle tissue, urine, and milk were sampled and analyzed using high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). The average daily intake of dairy cows was 3.4 ± 0.7, 4.6 ± 1.0, 7.6 ± 3.7 and 2.0 ± 1.2 μg/kg body weight (bw) for PFBS, PFHxS, PFOS, and PFOA, respectively. Overall, PFBS, PFHxS, PFOS, and PFOA showed different kinetics in dairy cows. In plasma, concentrations of PFBS (mean = 1.2 ± 0.8 μg/L) and PFOA (mean = 8.5 ± 5.7 μg/L) were low, whereas PFHxS and PFOS continuously increased during the PFAA-feeding period up to maximal concentrations of 419 ± 172 and 1903 ± 525 μg/L, respectively. PFOS in plasma remained constantly high during the depuration period. PFOS levels were highest in liver, followed by kidney, without significant differences between feeding periods. The highest PFHxS levels were detected in liver and kidney of cows slaughtered on day 29 (61 ± 24 and 98 ± 31 μg/kg wet weight (ww)). The lowest PFAA levels were detected in muscle tissue. At the end of the feeding study, cumulative secretion in milk was determined for PFOS (14 ± 3.6%) and PFHxS (2.5 ± 0.2%). The other two chemicals were barely secreted into milk: PFBS (0.01 ± 0.02%) and PFOA (0.1 ± 0.06%). Overall, the kinetics of PFOA were similar to those of PFBS and substantially differed from those of PFHxS and PFOS. The very low concentration of PFBS in plasma and milk, the relatively high urinary excretion, and only traces of PFBS in liver (0.3 ± 0.3 μg/kg ww) and kidney (1.0 ± 0.3 μg/kg ww) support the conclusion that PFBS does not accumulate in the body of dairy cows.