Palmitoleic acid, a monounsaturated fatty acid which could affect glucose and lipid metabolism and reduce insulin resistance has two isomers, i.e. cis-palmitoleic acid (cPOA) and trans-palmitoleic acid (tPOA). However, the pharmacokinetic, metabolic transformation and structure-activity relationship of the two isomers have not been reported. A precise and accurate ultra performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) method was developed to determine cPOA and tPOA simultaneously. Both the cPOA and tPOA were administered i.g. (intragastric gavage) to rats at 75 mg/kg. Serum samples were collected and analyzed for the two isomers by UPLC-MS/MS on a reverse-phase BDS C18 column equilibrated and eluted with water (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. The calibration curves for cPOA and tPOA were linear over the range 0.1-12 μg/mL. Analytes were monitored by selected-reaction monitoring in negative electrospray ionization mode. The Tmax of cPOA was 0.94 ± 0.44 h and the Cmax 8.17 ± 1.97 μg/L, and the Tmax and Cmax of tPOA were 1.50 ± 0.98 h and 14.77 ± 11.91 μg/L, respectively. AUC of cPOA and tPOA were 59.45 ± 29.83 and 113.88 ± 72.25 mg/L·h. The method was applied in pharmacokinetic study of cPOA and tPOA in rat serum successfully. Besides, the concentrations of cPOA and tPOA in rat serums were observed fluctuating with a consistent trend, which may be due to reciprocal bio-convert in the body.