The thyroid hormone disrupting activities of drinking water sources from the lower reaches of Yangtze River were examined using a reporter gene assay based on African green monkey kidney fibroblast (CV-1) cells. None of the eleven tested samples showed thyroid receptor (TR) agonist activity. Nine water samples exhibited TR antagonist activities with the equivalents referring to Di-n-butyl phthalate (DNBP) (TR antagonist activity equivalents, ATR-EQ 50 s) ranging from 6.92 × 10 1 to 2.85 × 10 2 μg DNBP/L. The ATR-EQ 50 s and TR antagonist equivalent ranges (ATR-EQ 30–80 ranges) for TR antagonist activities indicated that the water sample from site WX-8 posed the greatest health risks. The ATR-EQ 80 s of the water samples ranging from 1.56 × 10 3 to 6.14 × 10 3 μg DNBP/L were higher than the NOEC of DNBP. The results from instrumental analysis showed that DNBP might be responsible for the TR antagonist activities in these water samples. Water sources along Yangtze River had thyroid hormone disrupting potential.