Aims: TRPV1-expressing, capsaicin (CAP)-sensitive afferent fibers innervating bladder in addition to sensory function also exhibit efferent features consisting in TRPV1-dependent release of tachykinins (TAC) affecting detrusor smooth muscle (DSM) contractions. Our aim was to investigate the effects of two novel polypeptide inhibitors of TRPV1 from the venom of tropical sea anemone Heteractis crispa, APHC1 and APHC3, on the contractions of DSM from bladders of normal and diabetic rats. Main methods: Experiments were conducted on urothelium-devoid DSM strips from normal rats and rats 8 weeks after streptozotocin-induced diabetes by means of contraction force measurements. Key findings: Pre-exposure of DSM strips to APHC1 or APHC3 (200 nM) specifically inhibited CAP-induced, TRPV1-dependent contractions. Both peptides also transiently enhanced basal tone and spontaneous contractions of DSM strips followed by delayed suppression of electric field stimulation (EFS)-evoked nonadrenergic-noncholinergic (NANC) contractions. The decrease of the amplitude of EFS-evoked NANC contractions by APHC1 or APHC3 reached 38.5 ± 3.4% and 25.1 ± 1.6%, respectively, in normal DSM strips and 46.3 ± 3.3% and 43.9 ± 1.8%, respectively, in diabetic ones. APHC-peptide-induced transient enhancement of basal tone could be mimicked by serine protease inhibitor, 4-(2-aminoethyl)bezenesulfonyl fluoride (300 μM). Significance: Our results demonstrate that APHC1 and APHC3 may be considered as effective inhibitors of bladder contractility especially during diabetic cystopathy. Modality of action of APHC-polypeptides via the mechanisms involving decreased TRPV1-dependent release of TAC from bladder afferents and suppression of TAC degradation due to their activity as endogenous proteases inhibitors is proposed. © 2012 Elsevier Inc. All rights reserved.