Carboxylesterase 1 (CES1) is a major phase I drug-metabolizing enzyme, responsible for 80-95% of total hydrolytic activity in human livers. CES1 plays a crucial role in the metabolism of a wide range of drugs, pesticides, environmental pollutants, and endogenous compounds. The clinical relevance of CES1 has been well demonstrated in various clinical trials with methylphenidate, oseltamivir, and clopidogrel. Expression and activity of CES1 vary markedly among individuals, which is a major contributing factor to interindividual variability in the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs metabolized by CES1. The loss-of-function SNP G143E (rs71647871) is the only clinically significant CES1 variant identified to date. A multi-dose, prospective pharmacogenetics, pharmacokinetics-pharmacodynamics (PGx-PK/PD) clinical trial was conducted in healthy volunteers (n=21) to examine the impact of G143E on the PK and PD of a CES1 substrate, enalapril. Enalapril 10mg was given once daily for seven consecutive days to the G143E carriers (n=6) and the G143E non-carriers (n=15) prior to a 72h PK/PD study. The study found the CES1 G143E carriers had 30.9% lower enalaprilat Cmax (P = 0.03), 27.5% lower enalaprilat AUC0-∞ (P = 0.02), and 32.3% lower enalaprilat-to-enalapril AUC0-∞ ratio (P = 0.003) compared to the non-carriers. The average maximum reduction of systolic blood pressure (SBP) was used as the PD surrogate marker. The non-carrier group had 12.4% lower SBP at the end of the study compared to the baseline (P = 0.001), while no statistically significant SBP reduction was observed in the G143E carriers. The study confirmed the CES1 G143E variant significantly impacted the PK and PD of enalapril. G143E, however, can only explain a small portion of the interindividual variability in the CES1 function. A better understanding of the regulation of CES1 expression and activity could lead to the development of a precision pharmacotherapy strategy to improve the efficacy and safety of many CES1 substrate drugs. Here, we developed a novel allele-specific protein expression (ASPE) assay to identify genetic variants regulating CES1 protein expression in human livers. The ASPE method adopted a custom-designed heavy stable isotope-labeled QconCAT internal standard and utilized a nonsynonymous variant, S75N (rs2307240), as the marker to determine the allelic expression of CES1. Two cis-acting regulatory variants (rs6499788, rs35918553) were found to be associated with CES1 ASPE and protein expression in human liver microsome (HLM) samples (P < 0.05). The effects of the two variants were further validated by the CES1 protein expression and activity studies in human liver S9 fractions (HLS9) (P < 0.05). We expect that combining all identified functional CES1 variants (e.g., G143E, rs6499788, and rs35918553) will provide a more holistic view of CES1 pharmacogenetics and allow us to better predict the PK and PD of medications metabolized by CES1.