Abstract Background and purpose: 5-Hydroxytryptamine (5-HT) can enhance human ureteral contractions. However, the mediating receptors have not been clarified yet. The study sought to further characterize the mediating receptors using several more selective antagonists and agonists. Experimental approach: Human distal ureters were obtained from 88 patients undergoing cystectomy. The mRNA expression levels of 5-HT receptors were examined using RT-qPCR experiments. The phasic contractions of ureter strips, either spontaneous or evoked with neurokinin, were recorded in an organ bath. Key results: Among the 13 5-HT receptors, 5-HT2A and 5-HT2C had the highest mRNA expression levels. 5-HT (10-7–10-4 M) concentration-dependently increased the frequency and baseline tension of phasic contractions. However, a tachyphylaxis effect was observed. SB242084 (100 nM) and ketanserin (100 nM), which are 5-HT2C selective and non-selective antagonist, respectively, shifted the 5-HT concentration-response curves (frequency and baseline tension) rightward. 5-HT2C selective agonist, vabicaserin, increased contraction frequency with an Emax of 35% of 5-HT. 5-HT2A selective antagonist, volinanserin (100 nM), only reduced baseline tension. The selective antagonists of 5-HT1A,1B, 1D, 2B, 3, 4, 5, 6, and 7 had no antagonism. Blockade of voltage-gated sodium channels, α1-adrenergic receptors, adrenergic neurotransmission, and neurokinin-2 receptors using tetrodotoxin, tamsulosin, guanethidine, and Men10376, respectively, and desensitizing sensory afferents using capsaicin (100 μM), significantly reduced 5-HT effects. Conclusion and implications: 5-HT enhanced ureteral phasic contractions mainly by activating 5-HT2C. Activation of sympathetic nerve and sensory afferents partly contributed to 5-HT effects. 5-HT and 5-HT2C receptors could be promising targets for ureteral stone expulsion and ureteral colic relief.