We present the first record of Holothele longipes (L. Koch, 1875) (Araneae, Theraphosidae) from the Republic of Ecuador, based on a male specimen collected near Puerto Misahuallí, province of Napo, in the north-eastern foothills of the Andes, upper River Napo basin. This new record extends the geographic range of H. longipes by approximately 554 km west of the nearest previously known locality, on the River Momón, department of Loreto, Peru. Our new record also represents the first time a species of the theraphosid subfamily Ischnocolinae has been found in Ecuador.