Castrate-resistant prostate cancer remains a major clinical challenge. Due to the toxicity profile of taxane-based chemotherapy and treatment failure in some patients, novel agents with improved efficacy to side effect profiles are urgently needed. Eg5, a member of the kinesin-5 family, controls the formation of the bipolar spindle during cell division, and suppressed Eg5 function leads to mitotic arrest. S-Trityl-L-cysteine (STLC) is a novel Eg5-specific small-molecule inhibitor. Here, we report the first study to evaluate its use in prostate cancer. In a panel of prostate cancer cells, LNCaP and PC3 cells were the most and least sensitive to STLC treatment, with a 7.2-fold difference in their respective GI(50) values: 250 nmol/L and 1.8 micromol/L. In LNCaP cells, treatment with either STLC or docetaxel resulted in transient G(2)-M arrest and subsequent caspase-mediated cell death. However, STLC- and docetaxel-treated PC3M cells have distinct fates: STLC induced a transient G(2)-M arrest, followed by polyploidy; in contrast, docetaxel-treated PC3M cells progressed to apoptosis after a transient G(2)-M arrest. Docetaxel-resistant LNCaP-derived (LDocR) cells respond to STLC in a similar manner to the parental cells. Although the docetaxel-resistant PC3M-derived (PDocR) cell line and its parental PC3M cells have similar GI(50) to STLC treatment, PDocR cells showed significantly more G(2)-M arrest and less apoptosis. Hence, although docetaxel-resistant prostate cancer cells remain responsive to Eg5 inhibition with STLC, there are key differences at the cell cycle level, which may have implication in future development.