Interest in the exploitation of microalgae for biotechnological applications has increased over the last decade, and microalgae are now viewed as offering a sustainable alternative to traditionally used host chassis. A number of recombinant proteins have been expressed in genetically modified algal strains, with the green alga Chlamydomonas reinhardtii being a particularly popular host strain. While nuclear transformation is possible with this organism, chloroplast transformation offers more reliable expression, and several proteins have been expressed in the stroma. Here, we present the first utilisation of the thylakoid lumen for recombinant protein production in microalgae. A bacterial export signal peptide was used to efficiently translocate two recombinant proteins, a fluorescent reporter protein (pHRed) and a biopharmaceutical model substrate (scFv) into the thylakoid lumen. This approach expands the algal chloroplast genetic toolkit and offers a means of expressing proteins that are difficult to express in the stroma for reasons of toxicity, stability or a requirement for disulphide bonding.