Urethral smooth muscle (USM) contributes to urinary continence by contracting during the urine storage phase, which is mainly mediated by activation of post-junctional α1-adrenoceptors. Males and females show differences in the functioning of the lower urinary tract and the most common urinary tract symptoms (LUTS). LUTS in men typically occur in association with bladder outlet obstruction, whereas in women urinary urge-incontinence symptoms are more common. Therefore, this study aimed to evaluate sex differences in α1-adrenoceptor subtype expression and their importance in proximal urethra contraction in mouse (C57BL6/J) and marmoset (Callithrix jacchus). Contractile responses to phenylephrine, noradrenaline, potassium chloride (KCl) and electrical-field stimulation (EFS) were evaluated. Phenylephrine, noradrenaline, KCl and EFS produced markedly greater contractions in male mice and marmoset USM compared with females. The sex differences remained unchanged by L-NAME (NOS inhibitor), atropine (muscarinic antagonist) and PPADS (P2X1 purinoceptor antagonist). Additionally, selective α1A- (but not α1B- and α1D) adrenoceptor antagonists significantly reduced phenylephrine-induced USM contractions. qRT-PCR for α1A, B and C-adrenoceptor subtypes revealed a marked presence of α1A adrenoceptor subtype in male USM, but not females. Male mouse urethra also exhibited a higher tyrosine hydroxylase mRNA expression. Histomorphometric analysis showed a greater USM area in male than female mice. In conclusion, male mouse and marmoset proximal USM shows strong α1A adrenoceptor-induced contractions and abundant α1A adrenoceptor expression, whereas α1A adrenoceptor-mediated mechanisms are much less important in females. The differential expression of α1-adrenoceptors in the proximal urethra may contribute to the higher incidence of urinary incontinence in women and obstructed voiding in men.