Background: Long-chained poly- and perfluoroalkyl substances (PFAS) have been used in pesticide formulations but their potential impact on human PFAS exposure has not been addressed. Objectives: To investigate if occupationally pesticide exposed female greenhouse workers in Denmark had higher serum concentrations of PFAS than a comparable background population. Methods: Serum samples collected between 1996 and 2001 from 181 pregnant greenhouse workers and a contemporary urban population of 48 pregnant women were analyzed for eight PFAS: perfluorooctane sulfonate (PFOS), perfluorohexane sulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorooctane sulfonamide (FOSA), N-methyl perfluorooctane sulfonamido acetic acid (N-MeFOSAA), and N-ethyl perfluorooctane sulfonamido acetic acid (N-EtFOSAA). Results: The concentrations of PFOA, PFOS, and the PFOS precursors N-MeFOSAA, N-EtFOSAA, and FOSA were higher, and PFHxS was lower, among greenhouse workers than the comparison population. After adjusting for age and parity, serum concentrations of N-MeFOSAA, N-EtFOSAA, and FOSA were 2-to-3-fold higher, and the major PFAS in serum, PFOS and PFOA, were 30–50 % higher among the greenhouse workers. Conclusion: Higher serum concentrations of some legacy PFAS among female greenhouse workers indicate that exposure to pesticides is a potential pathway of exposure. Although PFAS use in pesticide applications may appear to be a minor source of exposure for the general population, this pathway deserves attention in risk assessment.