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• Horse manure (HM) was employed as a feedstock for bioethanol production. • High holocellulose contents in HM produced fermentable sugars with 80% yield. • N-rich HM showed competitive ethanol production (0.075 g L-1h−1) with no N support. Insecure supply chain of biomass has been regarded as one of the critical constraints for deteriorating the practical implementations of bioethanol (BE). To find a raw feedstock beyond lignocellulosic biomass, horse manure (HM) was converted into BE. To realize the grand premise, two pretreatment methods, acid-/alkaline-pretreatments using H 2 SO 4 /NaOH, were used for HM. To optimize acid-/alkaline-pretreatments, the surface methodology response with the Box-Behnken design was done. Under the optimized conditions, alkaline-pretreatment showed higher maximum sugar recovery yield (80%) than that from acid- pretreatment (71%), which offers that alkaline-pretreatment is suitable for BE synthesis from HM. The fermentability of acid/enzyme- and alkaline/enzyme-hydrolysates without a supplement of nitrogen source were tested using GRAS strain of yeast, Pichia stipitis. The results indicated that alkaline/enzyme- hydrolysates showed higher BE productivities (0.075 g L-1h−1) than those of acid/enzyme-hydrolysates (0.050 g L-1h−1). To elucidate the possible reasons of such higher BE productivities from the alkaline/enzyme- hydrolysates, the potential fermentative inhibitory compounds for Pichia stipitis such as acetic acid, furfural, and hydroxymethylfurfural were quantitatively analyzed. The results indicated that the presence of less amounts of toxic compounds from the alkaline/enzyme- hydrolysates may lead to such higher BE productivities as compared those present in acid/enzyme-hydrolysates. Also, all experimental results suggested a potential for saving production cost of BE using HM as the C/N sources without an additional nitrogen source supplement. [ABSTRACT FROM AUTHOR]