Fipronil, a widely used phenylpyrazole insecticide, is known to disrupt circulating thyroid hormone (TH) levels in rodents. Concentrations of fipronil and its metabolites (fipronil sulfone and fipronil sulfide) in serum samples collected in 2009–2011 were measured for 131 Japanese pregnant women by a sensitive and accurate liquid chromatography-tandem mass spectrometric method developed in our laboratory to relate TH levels. Fipronil sulfone was detected in all the subjects with the median being 21 ng/L (min–max: 6.8–89), but fipronil and fipronil sulfide were detected in none of the subjects (detection limit: 5.0 and 1.2 ng/L, respectively), indicating a rapid and exclusive oxidative conversion to fipronil sulfone upon exposure. The median concentration of fipronil sulfone was lower than those previously reported for general populations in other countries by one order of magnitude. There were no attributes or dietary habits of the subjects that significantly vary the serum fipronil sulfone concentrations. Multiple regression analyses found no significant association between serum concentrations of fipronil sulfone and free thyroxine- or thyroid-stimulating hormone levels, indicating the absence of adverse effects on circulating TH levels probably due to the lower exposure levels of the present subjects. The present result would be valuable for establishing a dose–effect relationship of fipronils in humans on population levels.