<p>Background: Cone snails are a natural source of complex peptides with analgesic properties called conotoxins. These peptides are secreted in a complex venomic mixture and are predominantly smaller than 5 <em>kDa</em>. The present study aimed to document the analgesic activity of two species of <em>Conus coronatus</em> (<em>C.</em> <em>coronatus</em>) and <em>Conus frigidus</em> (<em>C. frigidus</em>) venom collected off the Iranian coast in a mouse behavioral test.</p> <p>Methods: Conotoxin containing fractions was extracted from the venom ducts and initially purified by column chromatography. The analgesic effect of the fractions was determined on formalin pain model and hot-plate test.</p> <p>Results: The results led to the identification of four fractions with analgesic activity in <em>C. coronatus</em> and two in <em>C. frigidus</em>. Only one fraction was able to reduce the flinching and licking in both acute pain and chronic pain phases of the formalin test. Moreover, the activity of this fraction remained 30 minutes on the hot-plate test. Purification of the fractions was carried out by RP-HPLC. LC-ESI-MS analysis of the fractions showed that the conotoxins of the analgesic fraction had molecular weights not previously reported.</p> <p>Conclusion: The findings give insight into the venom of two previously under-investigated <em>Conus</em> species and reveal the therapeutic potential of the containing conopeptides.</p>