Riboflavin is an activator of defence responses in plants that increases resistance against diseases caused by fungal, oomycete, bacterial and viral pathogens. However, the mechanisms driving defence activation by riboflavin are poorly understood. We investigated the signal transduction pathways of phospholipase C (PLC) and phospholipase D (PLD) in tobacco (Nicotiana tabacum) suspension cells using a pharmacological approach to confirm whether riboflavin-mediated activation of the defence response is dependent on both PLC and PLD. The expression patterns analysed by quantitative reverse transcription-polymerase chain reaction demonstrated that the tobacco PLC and PLD gene families were differentially expressed in riboflavin-treated tobacco cells. PLC and PLD expression accompanied defence responses including the expression of defence response genes (PAL, PR-1a and PR-1b), the production of hydrogen peroxide and the accumulation of the phytoalexin scopoletin in tobacco cells treated with riboflavin. These defence responses were significantly inhibited in the presence of the PLC inhibitor U73122 and the PLD inhibitor 1-butanol; however, inhibitor analogues had no effect. Moreover, treating tobacco cells with phosphatidic acid, a signalling molecule produced by phospholipase catalysis, induced the accumulation of the phytoalexin scopoletin and compensated for the suppressive effects of U73122 and 1-butanol on riboflavin-induced accumulation of the phytoalexin. These results offer pharmacological evidence that PLC and PLD play a role in riboflavin-induced defences of tobacco.