Clonorchis sinensis thrives on bile juice. The effects of bile and bile acids on newly excysted juvenile C. sinensis (CsNEJ) were studied in terms of survival. Survival of CsNEJs maintained in 1x Locke's solution, Dulbecco's modified Eagle's medium, NCTC 109, Eagle's, RPMI 1640, and 0.1% glucose was high, but dropped rapidly in 2x Locke's, 0.85% NaCl, and phosphate-buffered saline. Most amino acids in the media favored CsNEJ survival; however, aspartic and glutamic acids and adenine reduced survival. Survival was also significantly lower in media containing more than 0.1% bile. CsNEJs preconditioned in low bile media survived longer in higher bile media. All bile acids and conjugated bile salts were found to favor CsNEJ survival, except for lithocholic acid (LCA) which was toxic. NCTC 109 medium was found to be optimal for the in vitro maintenance of CsNEJs and 1x Locke's solution to be suitable for analyzing the biological effects of bioactive compounds and molecules. Based on these results, we propose that bile acids enhance activity of CsNEJs, but LCA deteriorate CsNEJs.