Updated publication reference for PubMed record(s): 32152231. Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic activities. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, we find that androgen- induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo. Tissues from mice lacking ADTRP (Adtrp-KO), AIG1 (Aig1-KO), and AIG1/ADTRP (DKO) have higher concentrations of FAHFAs, including 9-PAHSA, due to decreased FAHFA hydrolysis activity. No other lipids were changing in these samples to indicate that AIG1 and ADTRP are specific FAHFA hydrolases. To complement these genetic studies, we identified a dual AIG1/ADTRP inhibitor, ABD-110207, that is active in vivo. Acute treatment of wild-type mice with ABD-110207 results in elevated FAHFA levels further supporting AIG1 and ADTRP enzymatic activity in controlling endogenous FAHFAs. The loss of AIG1/ADTRP does not mimic the effects of pharmacologically administered FAHFAs on glucose tolerance or insulin sensitivity in mice, indicating that therapeutic strategies should continue to focus on FAHFA administration. Together, these studies designate AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs.