Haematococcus pluvialis is a prominent feedstock of astaxanthin. The ratio of carbon to nitrogen (C/N) strongly influences the metabolic pathways of mixotrophic-grown microalgae, however, its role involved in astaxanthin biosynthesis is still not fully understood. In this study, integrative metabolic and physiologic profiles were analyzed in elucidating how C/N affected carbon and nitrogen assimilation and thereby exerted influence on astaxanthin biosynthesis. It was demonstrated that high C/N up-regulated the activities of acetate kinase by increase of 5.76 folds in early logarithmic phase, leading a significant increase of acetyl-CoA. The increased carbon skeletons were then funneled into astaxanthin biosynthesis. Additionally, high C/N increased the proportion of carotenoid-intermediates in cytoplasm from chloroplast. Finally, a fed-batch cultivation strategy based on C/N gradient was developed. Biomass attained 9.18 g L-1 in 100% type of immotile cyst cells, which presented astaxanthin productivity at 15.45 mg L-1 d(-1) afterward, exhibiting a promising paradigm in commercial production.