Non-structural carbohydrates (NSC) play an important role in yield formation. In this paper, the relationships of NSC accumulation and translocation with yield formation were investigated under elevated CO₂ concentrations ([CO₂]) and nitrogen (N) application rates. A japonica rice (Oryza sativa L.) cultivar, “Nanjing 9108,” was grown at three [CO₂]—Ambient (T0), ambient + 160 μmol·mol⁻¹ (T1), and ambient + 200 μmol·mol⁻¹ (T2)—in open-top chambers (OTC), with three levels of N fertilizer application rates—10 gN·m⁻² (N1), 20 gN·m⁻² (N2), and 30 gN·m⁻² (N3)—Which were set in OTCs using pot experiments. The results showed that the concentration of NSC (C_NSC) and the total mass of NSC stored in stems (TM_NSC) under T1 and T2 were higher than those in the T0 treatment, and the C_NSC and TM_NSC of N3 were lower than those of N1 and N2 at the heading stage. The C_NSC and the TM_NSC were significantly positively correlated with the stem biomass during the growth period and were notably negatively correlated with the N content in leaves (N_leaf) at the heading and filling stages. The seed setting rate was significantly positively related to the apparent transferred mass of NSC from stems to grains (ATM_NSC) at the filling stage, while the relationship between yield and the ATM_NSC was not statistically significant. Although there was no difference in the apparent contribution of transferred NSC to grain yield (AC_NSC) between treatments, NSC stored in stems further accumulated obviously during the late filling stage, implying that the grain yield of “Nanjing 9108” was predominantly sink-limited. We concluded that elevated [CO₂] improved the concentration of NSC at the rice heading stage. The interaction between elevated [CO₂] and N fertilizer rates significantly influenced the concentration of NSC at the filling stage. Rice stems NSC reaccumulated at the late grain filling stage, which implies a restriction on NSC transference to grain.