Pancreatic cancer (PC), a malignancy with very poor prognosis, presents many molecular alterations, including overexpression of Cyclin B1. However, the prognostic value of the protein in PC remains to be elucidated. In the present study, Cyclin B1 expression was detected immunohistochemically in specimens from 241 patients with PC and was correlated with clinicopathological features and patient survival. It was found that Cyclin B1 expression, located in nucleus and/or cytoplasm, was not statistically associated with clinicopathologic variables. However, overall survival of patients with high Cyclin B1 expression was significantly poorer than that of those with low Cyclin B1 expression (P = 0.010). Moreover, Cyclin B1 was identified as an independent prognostic factor by multivariate Cox regression test (P = 0.003). Finally, its independent implication for prognosis was proven in five subgroups of PC, i.e., males, patients aged ≤ 65 years, G1-2 and N0 tumors as well as those with perineural invasion (all P < 0.05). These data indicate that high expression of Cyclin B1 is a valuable molecular marker of unfavorable prognosis in PC.