Haematococcus pluvialis is a freshwater planktonic single-cell microalgae. It will accumulate high amount of carotenoids under unfavorable environmental conditions. As one of carotenoids, lycopene is an important intermediate in the carotenoid biosynthesis pathway. Lycopene β-cyclase (LycB) is the key enzyme that catalyzes the circularization of lycopene to form β-carotene. In this study, we constructed a p1301-BS-RNAi vector using lycB from Dunaliella salina as the interference sequence with kanamycin and atrazine resistance marker, and then transformed it into H. pluvialis by electroporation. Sixteen independent transgenic lines were obtained after resistance selection, genome PCR, and RT-PCR analyses. Seven well-grown lines were selected to determine the contents of carotenoids by HPLC analysis after inducing by high light. The lycopene content in these lines was increased by 99.4% while the β-carotene content was decreased by 48.4%, indicating that the interference by heterogenous lycB could inhibit the conversion of lycopene into β-carotene. The amount of increase in lycopene was only 5% of the amount of decrease in β-carotene, indicating that 95% of the decreased β-carotene was converted into other metabolites. Therefore, in order to largely increase the lycopene content in H. pluvialis, it is necessary to coordinatively regulate other metabolic pathways.