Interstitial cystitis is a chronic disease characterized by lower abdominal pain and some nonspecific symptoms including an increase in urinary frequency and urgency. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that controls smooth muscle tone via G-protein coupled receptors (S1P1–3 receptors). S1P production is known to take place both in physiological states and some pathological situations, such as in overactive bladder syndrome. The intracellular mechanism of S1P-induced contractile response was investigated in β-escin permeabilized detrusor smooth muscle of rats having cyclophosphamide-induced cystitis. The bladder was isolated from rats and detrusor smooth muscle strips were permeabilized with β-escin. S1P (50 μM)-induced contraction and calcium sensitization response were significantly increased in cystitis. S1P-induced augmented contractile response was inhibited by S1P2 receptor antagonist JTE-013 and S1P3 receptor antagonist suramin. S1P2 receptor protein expressions were increased in cystitis, where no change was observed in S1P3 expressions between control and cystitis groups. S1P-induced contraction was reduced by Rho kinase (ROCK) inhibitor Y-27632 and protein kinase C (PKC) inhibitor GF-109203X in both control and cystitis group. S1P-induced increased calcium sensitization response was decreased by ROCK inhibitor and PKC inhibitor in cystitis. Our findings provide the first evidence that interstitial cystitis triggers S1P-induced increase in intracellular calcium in permeabilized detrusor smooth muscle of female rats. Both S1P2 and S1P3 receptors are involved in S1P mediated enhanced contractile response. The augmentation in S1P-induced contraction in interstitial cystitis involves both PKC and ROCK pathways of calcium sensitization.