Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was previously shown as differentially expressed in granulosa cells (GC) of bovine pre-ovulatory follicles suggesting that JAK3 could modulate GC function and activation/inhibition of downstream targets. We used JANEX-1, a JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3, CDKN1B/p27 and MAPK8IP3/JIP3. Cultured GC were treated with or without FSH in the presence or not of JANEX-1. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited. Proliferation markers CCND2 and PCNA were reduced in JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1-treated GC. UHPLC-MS/MS analyses revealed phosphorylation of specific amino acid residues within JAK3 as well as CDKN1B and MAPK8IP3 suggesting possible activation or inhibition post-FSH or JANEX-1 treatments. We show that FSH activates JAK3 in GC, which could phosphorylate target proteins and likely modulate other signaling pathways involving CDKN1B and MAPK8IP3, therefore controlling GC proliferation and steroidogenic activity.