Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to generate ceramide, an important molecule involved in the regulation of various cellular responses. In this study, we partially purified the neutral sphingomyelinase2 (nSMase2) and identified the inhibitors, D-lyxophytosphingosine and D-arabino-phytosphingosine, which have an inhibitory effect on nSMase2 in a concentration-dependent manner. A Dixon plot of each phytosphingosines revealed their probable inhibitory pattern, i.e., apparent competitive inhibition. These compounds did not inhibit the $Mg^{2+}$-independent neutral SMase activity, although the known nSMase2 inhibitor, GW4869, showed inhibitory effects on $Mg^{2+}$-independent neutral SMase activity. Further, the two phytosphingosines specifically inhibited the ceramide generation regulated by nSMase2.