Background Rice amylose content and amylopectin structure corporately determine rice eating and cooking qualities (ECQs).Soluble starch synthase(SS)IV-2is a member of the soluble starch synthesis gene family but with unknown effects on ECQs. Results In this study, three populations derived from a cross of two parents who possess the same major genes of starch bio-synthesis were employed to investigate the influence ofSSIV-2and its combined effects withADPglucose pyrophorylase large unit(AGPlar) andPullulanase(PUL) on ECQs. The results illustrated that the polymorphism ofSSIV-2alleles significantly affected gel consistency (GC), gelatinization temperature (GT), percent of retrogradation (PR) and three crucial rapid viscosity analysis (RVA) profile parameters: peak viscosity (PKV), breakdown viscosity (BDV) and setback viscosity (SBV). AndSSIV-2allele derived from CG173R had better quality traits with lower GT, SBV and PR. Moreover, its interaction withAGPlarwas responsible for the variations of GC, apparent amylose content (AAC), GT, PR and all RVA parameters except for pasting temperature (PaT) and peak time (PeT), in terms of GC, PKV and CSV,AGPlarderived from CG173R had an epistatic effect onSSIV-2; additionally, interaction ofSSIV-2andPULmainly affected GC, AAC, PKV, CPV, CSV and SBV. I-C and C-1 (I, allele ofAGPlarfrom Guangzhan 63S; C, allele ofSSIV-2from CG173R; 1, allele ofPULfrom Guangzhan 63S) combinations had better ECQs. Conclusions SSIV-2alleles significantly affect rice quality, especially the parameters relevant to gelatinized and thermal characteristics of starch (GC, PR, GT, PKV, BDV and SBV) under the same major genes (WaxyandSSII-3) background. It indicates thatSSIV-2functions elongation of starch chain. These findings suggest that the effects ofSSIV-2and its interaction withAGPlarandPULare vital for rice quality breeding with the same major genes.