Photobiological hydrogen production by green algae, such as Chlamydomonas reinhardtii is an attractive approach to generate renewable energy, but is currently not economically viable. It has been suggested that hydrogen yields could potentially be improved by eliminating or down-regulating competing fermentative pathways. However, at present fermentative metabolism is not completely understood in C. reinhardtii, such as the nature of the ill-defined D-lactate dehydrogenase (D-LDH) activity responsible for the production of D-lactate. To characterise the D-LDH activity, a bioinformatics analysis identified a candidate nucleus-encoded D-LDH in the C. reinhardtii genome (Phytozome v9.1 ID: Cre07.g324550), which was predicted to be localised to the chloroplast. The putative protein without its predicted chloroplast transit peptide was overexpressed in Escherichia coli as a C-terminal His6-tagged protein to confirm its function and assess its structure. Enzyme assays confirmed that the protein was an NAD+-dependent D-LDH, favouring the reduction of pyruvate to D-lactate with an estimated Km value of 1.85 ± 0.05 mM and kcat value of 415 ± 18 s-1. Size-exclusion chromatography suggests the holoenzyme was tetrameric with a molecular mass of about 202 kDa. Artificial microRNA technology was used to reduce the amount of D-LDH protein to less than 20% of WT levels. D-lactate was still produced in the mutant either from residual D-LDH activity or from other routes such as from methylglyoxal. Both NMR and HPLC confirmed that the knockdown did not have any substantial impact on dark anaerobic metabolite production except in slightly increasing the pyruvate levels. Additionally, the knockdown did not improve hydrogen yields under sulphur-deprived conditions. To assess the impact of eliminating fermentative pathways on metabolism, a series of mutants was isolated through cell mating, targeting the enzymes D-LDH, pyruvate decarboxylase (PDC3), pyruvate formate lyase (PFL1) and a bifunctional acetaldehyde/alcohol dehydrogenase (ADH1). Dark anaerobic metabolite production in the quadruple mutant confirmed the re-routing of metabolic flux from ethanol and formate towards glycerol and D-lactate. There was also a reduced flux towards acetate production. Pyruvate and glucose levels were found to be elevated in this mutant. Gas chromatography analysis suggested that downregulation of the fermentative pathways did not improve hydrogen production under sulphur-deprived conditions, in part because of reduced cell viability. These mutants are promising tools for future studies probing the metabolism of C. reinhardtii.