8-(3-chlorostryryl) caffeine (CSC), a selective adenosine A(2A) receptor antagonist, has been reported to inhibit the levodopa-induced motor fluctuation in Parkinson's disease. However, the underlying mechanism of its action remains largely unknown. In our study, we investigated the signaling pathway by which CSC inhibited levodopa-induced motor fluctuation in rats with a 6-hydroxydopamine (6-OHDA)-induced lesion. We treated 6-OHDA-lesioned rats with levodopa (50 mg/kg/day, twice daily) for 22 days, followed by levodopa+CSC (5 mg/kg/day, twice daily) or levodopa+vehicle for 7 days. The sham-lesioned and 6-OHDA-lesioned rats treated with saline for 29 days served as sham and lesion control groups. We found that the treatment of CSC reversed the shortening of the rotational motor response duration induced by levodopa administration and the effect was maintained until the end of the treatment. The chronic levodopa treatment upregulated the adenosine A(2A) receptor expression and modified downstream signaling pathway including decreasing the phosphorylation of DARPP-32 at Thr75 site and increasing the phosphorylation of ERK1/2 in the lesioned striatum. However, the following CSC treatment attenuated the levodopa-induced adenosine A(2A) receptor upregulation and abolished the aberrant phosphorylation of DARPP-32 at Thr75 site and that of ERK1/2. Our results indicate that the inhibitory effect of CSC on levodopa-induced motor fluctuation may be associated with the inhibition of Adenosine A(2A) Receptor and downstream DARPP-32 and ERK1/2 signaling pathway.