Fluorotelomer alcohols [FTOHs, F(CF2)nCH2CH2OH, n = 4, 6, and 8] are emerging environmental contaminants. Biotransformation of FTOHs by mixed bacterial cultures has been reported; however, little is known about the microorganisms responsible for the biotransformation. Here we reported biotransformation of FTOHs by two well-studied Pseudomonas strains: Pseudomonas butanovora (butane oxidizer) and Pseudomonas oleovorans (octane oxidizer). Both strains could defluorinate 4:2, 6:2, and 8:2 FTOHs, with a higher degree of defluorination for 4:2 FTOH. According to the identified metabolites, P. oleovorans transformed FTOHs via two pathways I and II. The pathway I led to the production of x:2 ketone [dominant metabolite, F(CF2)xC(O)CH3; x = n − 1, n = 6 or 8], x:2 sFTOH [F(CF2)xCH(OH)CH3], and perfluorinated carboxylic acids (PFCAs, perfluorohexanoic, or perfluorooctanoic acid). The pathway II resulted in the formation of x:3 polyfluorinated acid [F(CF2)xCH2CH2COOH] and relatively minor shorter-chain PFCAs (perfluorobutyric or perfluorohexanoic acid). Conversely, P. butanovora transformed FTOHs by using the pathway I, leading to the production of x:2 ketone, x:2 sFTOH, and PFCAs. This is the first study to show that individual bacterium can bio-transform FTOHs via different or preferred transformation pathways to remove multiple CF2 groups from FTOHs to form shorter-chain PFCAs. Biotechnol. Bioeng. 2012; 109: 3041–3048. © 2012 Wiley Periodicals, Inc.