We describe adults, tadpoles, advertisement calls, and provide information on the natural history of a new Amazonian species of Colostethus from Rio Branco, Acre, Brazil. The new species is diagnosed by small size, sexual dimorphism in ventral coloration, absence of median lingual process, white throat in males, testis white in adult males, absence of dorsolateral and ventral stripes, terminal discs of digits swollen, absence of swollen Finger III, fringes and webs absent on fingers, rudimentary interdigital webs between Toes III and IV. The species also differs from other described Colostethus species in tadpole morphology and acoustics. This species is the first Colostethus known to lay its eggs on the lower surface of leaves. Clutches of C. subfolionidificans, new species, are composed of about six to eight white eggs and are attended by the male. The tadpoles develop on the lower surface of leaves until stage 25, when they are carried by the male to complete metamorphosis in pools.

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