Anther culture is one of the most efficient methods for producing haploid and doubled-haploid plants to accelerate breeding programs in sunflower. This study was conducted to investigate the interaction effect of inoculating mother plants with the fungus Serendipita indica and different concentrations of the growth regulators 2,4-D and BAP on callus induction and regeneration from anther culture of three sunflower cultivars (Barzegar, Farrokh, and Ghasem) using a factorial experiment based on a completely randomized design. Sunflower seeds were inoculated with S. indica in the field, and after confirming symbiosis, buds at the R2 stage (1.5–2 cm diameter) were harvested. Anthers were cultured on solid MS basal medium containing various combinations of 2,4-D (0, 1, 2, 5 mg L⁻¹) and BAP (0, 0.5, 1 mg L⁻¹), and the resulting calli were transferred to three different organogenesis media containing NAA, BAP, kinetin, and IAA. The results showed that the main effect of the fungus on callus induction percentage was not significant (P>0.05), but the three-way interaction of fungus, BAP, and 2,4-D on callus quality was significant at the 1% level. The best combination for callus induction was 1 mg L⁻¹ BAP plus 1 mg L⁻¹ 2,4-D, which yielded an average of 75% callus induction. Furthermore, in fungus-inoculated plants, callus quality in this treatment (mean 2.2) was significantly better than in control plants (mean 1.3). Maximum regeneration (approximately 61%) was observed on MS medium containing 2 mg L⁻¹ BAP and 1 mg L⁻¹ NAA. In conclusion, the use of mother plants inoculated with S. indica in combination with optimized growth regulators can improve the quality of calli derived from sunflower anther culture.