Abstract Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.