Alpha/beta-hydrolase domain 6 (ABHD6) has recently been demonstrated to hydrolyze the endogenous cannabinoid. To examine the physiological and pathological roles of ABHD6 in vivo, we studied the phenotypic examination of Abhd6-knockout rats created by CRSPR/Cas9. Abhd6^−/− and the age-matched wild type (WT) rats of both sexes were used. Expression of Abhd6, assessed by RT-qPCR and Western blot analysis, obviously diminished in Abhd6^−/− rats compared with WT rats. Mutant rats generally looked normal in appearance, and the body weight and food intake were similar with WT rats. Locomotor activity of 24 hr and light phase significantly increased in Abhd6^−/− rats compared with WT rats (P<0.05). The interval between bladder contractions accessed by continuous cystometry was significantly shorter in Abhd6-knockout rats than WT rats (P<0.01) when the bladder was stimulated with acetic acid. Mechanical paw withdrawal threshold measured by von Frey test significantly lowered in Abhd6^−/− rats compared with WT rats (P<0.01). Oral administration of various analgesics raised the withdrawal threshold in Abhd6^−/− rats. We firstly demonstrate that deletion of Abhd6 gene in rats causes increased locomotor activity, frequent urination in stimulated bladder and hyperalgesia to non-noxious mechanical stimuli.