The impact of a deficiency in natural anticoagulants on the occurrence of cerebral venous thrombosis (CVT) is controversial, as well as whether there is a difference between CVT and deep venous thrombosis (DVT). The present study aimed to evaluate the association between a deficiency in natural anticoagulants and the occurrence of CVT vs. DVT. For this purpose, 274 patients newly diagnosed with venous thromboembolism (VTE), including 114 patients with DVT (41.6%), 81 patients with CVT (29.6%) and 79 patients (28.8%) with another type of VTE were retrospectively analyzed. In addition, 219 patients without thrombosis were used as the control group. Protein C (PC), protein S (PS) and antithrombin III (AT III) assays were performed prior to commencing treatment. The rates of PC, PS, AT III deficiency in the VTE group were 23.7, 28.8 and 14.2%, respectively. The rates of PC, PS, AT III deficiency in the CVT group were 21, 29.6 and 7.4%, respectively. The rates of PC, PS, AT III deficiency in the DVT group were 28, 34.2 and 15.8%, respectively. There was no significant difference between the DVT and CVT groups. Univariable and multivariable regression analysis revealed that PS deficiency was associated with the occurrence of all VTE types, DVT and CVT with odds ratios of 1.895, 2.330 and 2.052, respectively. On the whole, the present study demonstrates that PS deficiency is associated with the occurrence of CVT. No marked differences were noted between the deficiency in natural anticoagulants and CVT and DVT. These results may prove to be useful in deciding whether to perform natural anticoagulants testing in patients with CVT.