WsMAGO2 a duplicated protein in Withania through interactions with MPF2-like proteins affects male fertility by producing fewer flowers and aborted non-viable pollens/seeds regulated by anther-specific GAATTTGTGA motif. The MAGO NASHIs are highly conserved genes that encode proteins known to be involved in RNA physiology and many other developmental processes including germ cell differentiation in animals. However, their structural and functional implications in plants as fertility function proteins remained fragmented. MAGO (shorter name of MAGO NASHI) proteins form heterodimers with MPF2-like MADS-box proteins which are recruited in calyx identity and male fertility in Solanaceous plants. Four MAGO genes namely WsMAGO1 and WsMAGO2 and TaMAGO1 and TaMAGO2 were isolated from Withania somnifera and Tubocapsicum anomalum, respectively. These genes have duplicated probably due to whole genome duplication event. Dysfunction of WsMAGO2 through double-stranded RNAi in Withania revealed suppression of RNA transcripts, non-viable pollens, fewer flowers and aborted non-viable seeds in the developing berry suggesting a role of this protein in many traits particularly male fertility. WsMAGO2 flaunted stronger yeast 2-hybrid interactions with MPF2-like proteins WSA206, WSB206 and TAB201 than other MAGO counterparts. The native transcripts of WsMAGO2 culminated in stamens and seed-bearing berries though other MAGO orthologs also exhibited expression albeit at lower level. Coding sequences of the two orthologs are highly conserved, but they differ substantially in their upstream promoter regions. Remarkably, WsMAGO2 promoter is enriched with many anther-specific cis-motifs common in fertility function genes promoters. Among them, disruption of GAATTTGTGA abolished YFP/GUS gene expression in anthers alluding towards its involvement in regulating expression of MAGO in anther. Our findings support a possible recruitment of WsMAGO2 in fertility trait in Withania. These genes have practical application in hybrid production through cytoplasmic male sterility maintenance for enhancement in crops yield.