The role of sphingomyelin synthase 1 (SMS1), the Golgi membrane isoform of the enzyme, in ceramide metabolism and apoptosis after photodamage with the photosensitizer Pc 4 (PDT) is unclear. In the present study, using electrospray ionization/double mass spectrometry, we show that in Jurkat cells overexpressing SMS1, increases in ceramides were lower than in empty-vector transfectants post-PDT. Similarly, the responses of dihydroceramides and dihydrosphingosine, precursors of ceramide in the de novo synthetic pathway, were attenuated in SMS1-overexpressor after photodamage, suggesting the involvement of the de novo pathway. Overexpression of SMS1 was associated with differential regulation of sphingomyelin levels, as well as with the reduced inhibition of the enzyme post-treatment. Concomitant with the suppressed ceramide response, PDT-induced DEVDase activation was substantially reduced in SMS1-overexpressors. The data show that overexpression of SMS1 is associated with suppressed ceramide response and apoptotic resistance after photodamage.