Ras-Raf-MAPK signaling promotes cell proliferation and cell survival. We previously reported that Ras1CA overexpression, specifically in the posterior silk glands (PSGs) of the silkworm Bombyx mori, increased fibroin synthesis and cell size, resulting in improved silk yields. In this study, we compared the iTRAQ-based phosphoproteomic profiles of PSGs from wild-type and Ras1CA-overexpressing silkworms. Silk gland factor 1 (SGF1), a FOXA transcription factor that plays a critical role in activating fibroin gene expression, was identified as a phosphoprotein harboring Ser91 as a potential MAPK phosphorylation site. Ser91 phosphorylation of SGF1 was enhanced by Ras1CA overexpression, and this finding was verified by selected reaction monitoring. Consistently, MAPK activity is well correlated with Ser91 phosphorylation of SGF1 and its nuclear localization in PSG cells during silkworm development. Ras1CA overexpression and treatment with inhibitors of Ras signaling promoted or inhibited SGF1 nuclear localization, respectively; mutation of Ser91 to Ala91 eliminated SGF1 nuclear localization. Moreover, MAPK binds to SGF1 and directly phosphorylates Ser91, demonstrating Ser91 as a MAPK phosphorylation site in SGF1. In conclusion, Ras-Raf-MAPK signaling promotes SGF1 nuclear localization for transactivation via Ser91 phosphorylation in silkworms, showing that FOXA transcription factors are regulated via MAPK phosphorylation in animals.