Transient Receptor Potential (TRP) cation channels as TRP Vanilloid 1 and TRP Ankyrin 1 (TRPV1 and TRPA1) are nocisensors playing important role to trigger pain. These cation channels serve as thermosensors and several exogenous and endogenous chemical ligands activate them. Capsaicin and resiniferatoxin (RTX) activate TRPV1, allyl‐isothiocyanate (AITC) and formaldehyde activate TRPA1 receptors. Lipid rafts are defined as liquid ordered plasma membrane microdomains rich in cholesterol, sphingomyelin and gangliosides. Sphingomyelinase (SMase) decreases membrane sphingomyelin by hydrolization, myriocin inhibits the glycosphingolipid synthesis. Our aim was to examine their in vitro actions on TRP channels, and the potential analgesic effect of SMase and myriocin in in vivo mouse models.The effect of SMase and myriocin was analysed on isolated trigeminal (TG) neurones by measuring agonists‐induced Ca2+ transients with ratiometric technique, and on TRPV1‐ or TRPA1‐expressing CHO cells by measuring 45Ca‐uptake. Mechanonociceptive and thermonociceptive thresholds of the animals in RTX‐induced thermal, and mechanical hyperalgesia, and formaldehyde‐evoked hyperalgesia model were measured. The analgesic effect of SMase and myriocin was measured in capsaicin‐evoked acute nocifensive response test.It has been revealed that intracellular Ca2+ enhancement evoked by capsaicin (TRPV1), AITC and formaldehyde (TRPA1) was inhibited after SMase and myriocin incubation in TG neurons and receptor‐expressing cells. Treatment with SMase and myriocin significantly diminished the RTX‐induced thermal, and mechanical hyperalgesia, the formaldehyde‐evoked hyperalgesia and the capsaicin‐evoked nocifensive reaction.On the basis of all these findings it can be concluded that the lipid membrane environment modulates the function of TRPV1 and TRPA1 channels and lipid raft interfaces modulate the channel opening. Targeting this interaction might be a tool for drug developmental purposes.Support or Funding InformationJános Bolyai Fellowship, KTIA_NAP_ 20017‐1.2.1‐NKP‐2017‐00002, GINOP‐2.3.2‐15‐2016‐00050, EFOP‐3.6.2‐16‐2017‐00008, 20765/3/2018 FEKUTSTRAT