Increased levels of prolactin often coincide with an increased risk for thromboembolic events, but it is unclear whether a direct causal relation exists. Our aim was to examine the effect of prolactin on platelet function. In addition to using recombinant prolactin for experiments in vitro, we analyzed platelet function by flow cytometry in a group of 13 females with hyperprolactinaemia and 18 healthy female controls. Platelet activation was measured by P-selectin expression and by the amount of platelet-bound fibrinogen after stimulation with adenosine di phosphate (ADP), collagen-related peptide and the protease activated receptor (thrombin receptor) (PAR)-activating peptides PAR4-AP and PAR1-AP. Free oscillation rheometry was used to measure clotting time in whole blood. No significant effect on platelet activation or clotting time could be seen in in vitro experiments by adding recombinant prolactin. However, significantly lower P-selectin expression was found in the hyperprolactinemic group when platelets were activated by ADP (5 and 10 μM) or PAR4-AP. The expression of fibrinogen did not differ between the two groups for any of the activators used. For all samples, inverse significant correlations between P-selectin expression and prolactin concentration were found for both 5 μM ADP (r = - 0.61, p < 0.01), 10 μM ADP (r = - 0.62, p < 0.001) and PAR4-AP (r = - 0.69, p < 0.001). Thrombin cleavage of recombinant prolactin resulting in a 16 kDa C-terminal fragment did not alter the P-selectin expression upon activation. We found an indirect inhibitory effect of prolactin on platelets in hyperprolactinemic patients, suggesting that prolactin might have a protective role in thromboembolic disease.