Symbiosis has been associated with evolutionary innovations. For instance, the acquisition of nutritional, obligate ‘primary’ endosymbionts (P-symbionts) allowed insects to feed on plant sap and colonize novel ecological niches through the provision of nutrients lacking in their diet. P-symbionts often co-exist with facultative ‘secondary’ endosymbionts (S-symbionts) which may also contribute to their hosts’ niche specificity. The whitefly Bemisia tabaci is a major economic pest and a highly diversified species complex. In addition to the P-symbiont Portiera aleyrodidarum, most individuals harbor S-symbionts, whose role are not fully elucidated. This work addressed the fundamental question of whether and how S-symbiont composition influences the host plant exploitation by investigating the phenotypic and metabolic responses of three B. tabaci lines with different cytotypes and reared on three plants, hibiscus, lantana, or tobacco. We first analyzed the free amino acid composition of these three plants, and showed that they constitute a nutritionally contrasted environment. We then demonstrated that fecundity and free amino acid profiles differ when insects are reared on different plants, suggesting an important influence of the plant nutritional properties on these parameters. Lantana was the less favorable plant and was associated with a significant reduction in insect fecundity. Additionally, whiteflies on lantana showed a specific amino acid profile and thus a different physiological state when compared to individuals reared on the other plants. We also found fecundity differences between insect lines on lantana. To determine whether these differences rely on nuclear or extranuclear determinants, we produced genetically homogeneous hybrids, differing in their cytotype (the insect cytoplasmic features, including the intracellular symbionts) and demonstrated a clear contribution of the cytotype in determining female fecundity and amino acid profile on lantana. As the cytotypes used here differed in their S-symbionts composition, we propose that S- symbionts can contribute to the niche specificity and diversification of whiteflies.