The effects of CO2 enrichment on the in vitro growth and acclimatization of Protea cynaroides L. plantlets were investigated. Three CO2 enrichment concentrations were used: 0 (control), 1000, 5000, and 10000 μmol·mol−1. Plantlets in the control treatment were cultured on half-strength Murashige and Skoog (MS) medium supplemented with sucrose, whereas those enriched with different CO2 concentrations were grown on sucrose-free MS medium. Compared with the control, significant improvements were observed in the growth of plantlets enriched with CO2 irrespective of the concentration. Plantlets enriched with 5000 μmol·mol−1 CO2 produced the highest number of leaves and the largest leaf area. In addition, the photosynthetic ability of plantlets enriched with CO2 was enhanced, which resulted in significant increases in shoot growth and dry matter accumulation. In particular, the shoot dry weight of plantlets cultured in 5000 μmol·mol−1 CO2 and 10000 μmol·mol−1 CO2 were, respectively, 2.1 and 4.2 times higher than those without CO2 enrichment. During acclimatization, the survival percentage, rooting percentage, and leaf number of plantlets grown in elevated CO2 were, respectively, up to 4.5, 1.8, and 2.7 times higher than plantlets without CO2 enrichment. The improvements in survival percentage and ex vitro growth of these plantlets were the result of their enhanced photosynthetic ability in vitro, which resulted in the production of high-quality plantlets. Significant improvements in the overall growth of P. cynaroides plantlets were achieved through the use of photoautotrophic micropropagation with CO2 enrichment.