<p>Homeostasis of inorganic phosphate (P_i) in the chloroplasts is essential for healthy CO₂ assimilation. When P_i in chloroplasts is insufficient, the increase in the CO₂ assimilation rate (<i>A</i>) with an increase in CO₂ level is restricted, whereas <i>A</i> per unit total protein level moderately decreases under low-to-normal CO₂ levels. Some phosphate transporters (PHT) are localized in the chloroplast envelope; however, their contribution to the maintenance of P_i homeostasis for CO₂ assimilation has rarely been reported. In this study, we generated transgenic rice plants with RNAi-suppressed <i>PHT4;4</i>, one of the two genes of chloroplast envelope-localized PHT, and examined the changes in the characteristics of CO₂ assimilation. In three transgenic lines, the mRNA levels of <i>PHT4;4</i> decreased by approximately 80% without a notable decrease in total leaf-P levels or total leaf-N levels, which is thought to approximately correspond to total protein levels. <i>A</i> in the transgenic plants tended to be slightly lower than that in the wild-type plants, irrespective of the CO₂ level, and typically increased to saturation with increasing CO₂ levels. <i>A</i> per unit total leaf-N level in transgenic plants tended to be slightly lower than that in wild-type plants. These results indicate that substantial <i>PHT4;4</i> suppression caused slight symptoms of P_i-limited CO₂ assimilation. Therefore, PHT4;4 is suggested to be involved in the maintenance of chloroplast P_i homeostasis for healthy CO₂ assimilation, although its contribution is minor.</p>