A new anticancer ligustrazine derivative, 3beta-hydroxyolea-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (T-OA, C38H58O3N2), was previously reported. It was synthesized via conjugating the effective antitumor ingredients of a classic traditional Chinese medicine (TCM) formulation. In the present study, anticancer efficacy of T-OA was evaluated in vivo using a murine sarcoma S180 model. Reduction of the tumor weight and tumor HE staining regions demonstrated that T-OA had promising inhibition effects and a 50% inhibitory rate in S180 mice. Combining the immunohistochemistry, we found T-OA exerted its antitumor activity by preventing the expression of nuclear transcription factor NF-kappaB/p65 and COX-2 in S180 mice. The acute toxic test showed that LD50 value of T-OA exceeded 6.0 g/kg via gavage in mice. In addition, a simple and rapid HPLC-UV method was developed and validated to study the pharmacokinetic characteristics of the compound. After single-dose oral administration, time to reach peak concentration of T-OA (3.97 microg/mL) was 8.33 h; the elimination half-life and area under the concentration-time curve from t = 0 to the last time of T-OA was 4.50 h and 48.01 microg x h/mL, respectively.