The present study was aimed at understanding the possible association of allene oxide synthase (AOS), an enzyme implicated in the octadecanoid pathway during the pearl millet-downy mildew interaction. AOS 13-HPOT (13-hydroperoxy-9,11,15-octadecatrienoic acid) metabolizing activity assays assessed in various pearl millet cultivars with differential resistances against downy mildew revealed a positive correlation between cultivar resistance levels and AOS activities. Furthermore, the involvement of AOS in response to downy mildew was demonstrated by induction of AOS activity in both susceptible and resistant pearl millet cultivars during Sclerospora graminicola infection with higher induction observed in the resistant cultivar. Consistently, western blot analysis and tissue-blot immunoassay demonstrated the remarkable increase in AOS protein accumulation in the incompatible interaction. In addition, the tissue-blot immunoassay also showed the compartmentalization of AOS in the epidermis and vascular bundles of pearl millet seedlings. Expression analysis of a putative PgAOS1 gene revealed a marked difference in accumulation of PgAOS1 transcripts between contrasting plants, with pathogen-induced higher accumulation of the transcripts observed only in the resistant cultivar; a result which is in agreement with pathogen-induced AOS level and activity, indicating that PgAOS1 plays an important role in regulation of AOS level and activity in pearl millet upon S. graminicola infection. Our findings suggest an important role for AOS in regulation of responses to downy mildew disease in pearl millet. The differential AOS activities can potentially be used for selection of new disease-resistant pearl millet varieties, and the identified AOS-encoding gene(s) as genetic resource for development of enhanced downy mildew-resistant cultivars.