The number of seedlings per hill and plant spacing are critical determinants of high-yield rice (Oryza sativa L.) crops, yet their specific impacts remain unclear. This study examined four rice varieties in a two-year field experiment in Nanning and Yulin, Guangxi, using direct seeding and transplanting methods with a baseline seedling density of 60×10 4 ha -1. Biomass, nitrogen, and organic carbon accumulation in different plant organs were measured at maturity. The experimental location and season were treated as random effects, while planting methods, varieties, and seedlings per hill (spacing) were fixed effects. Linear models, linear mixed-effects models, ridge regression, and structural equation modeling were employed to assess the influence of seedling per hill and spacing on rice growth, biomass distribution, nitrogen and carbon accumulation, and yield. Results showed that increasing seedlings per hill and plant spacing reduced yield and above-ground biomass but increased the harvest index (HI). Single seedlings with narrow spacing produced the highest yield (7.27 t ha -1), above-ground biomass (16.4 t ha -1), and the lowest HI (0.46). Seedling number per hill and plant spacing negatively affected tiller count, effective panicles, leaf area index, and nutrient accumulation, while positively influencing specific leaf area and panicle biomass allocation. Organic carbon accumulation (40.04%) and nitrogen accumulation (22.34%) were major contributors to yield variation, with organic carbon alone explaining 50.9% of HI variation. Path analysis indicated that 96% of yield variance was accounted for, highlighting the importance of panicle biomass, organic carbon accumulation, and the source-sink balance between stems and panicles. In conclusion, single seedlings with narrow spacing optimize source-sink dynamics, enhancing biomass production and yield by compensating for lower material conversion efficiency.