Abstract: Objective To study the pharmacokinetic profile of phentolamine mesylate injection in healthy Chinese volunteers. Methods A total of 16 healthy volunteers were randomly divided into two groups, each receiving anterior teeth submucosal infiltration anesthesia and inferior alveolar nerve block anesthesia, respectively. The participants were injected with 0.9 mL, 1.8 mL, and 3.6 mL of 2% lidocaine HCl with 1∶100 000 epinephrine over three periods sequentially, followed by corresponding sequential injection of 0.2 mg, 0.4 mg, 0.8 mg of phentolamine mesylate at the same sites 30 min later.Blood samples were drawn from 5 min before injection to 15 h post the injection of phentolamine mesylate (16 time points). Adverse events were closely observed all the time. Plasma phentolamine mesylate was detected using UPLC-MS/MS with isotope as internal standard. WinNolin 6.1 software was used to calculate the pharmacokinetic parameters. Results Time to peak concerntration (t_max) ranged from 12 to 13 min. Half-time of elimination (t_1/2) ranged from 3.84 to 4.07 h, with a clearance (CL) of 190 L/h. Peak concentration (C_max), area under concentration-time curves from 0 to t hour and from 0 to infinite time (AUC_0-t and AUC_0-∞) increased proportionally in the dose range of 0.2 mg to 0.8 mg. The results of confidence interval analysis showed nearly linear dynamic characteristics for the injection of phentolamine mesylate. All participants experienced mild adverse events, including pain at the injection point, dizziness, and palpitations. These adverse events disappeared without treatments. Conclusion Phentolamine mesylate injection is effective for reversing oral local anesthetic effects.