Eradication of tumours by photodynamic therapy (PDT) is accompanied by marked changes in local sphingolipid (SL) engagement. Because of the heterogeneity of cellular composition, analysis of tumour tissue homogenates to quantify SL species is inadequate for evaluating their levels in parenchymal cancer cell population. By staining tumour-derived single cell suspensions with antibodies specific to ceramide and sphingosine 1-phosphate (S1P) followed by flow cytometry, we were able to document changes in the levels of these two key SLs in cancer cells and tumour-associated macrophages (TAMs) of mouse SCCVII tumours following PDT. The results confirm previously obtained indications that tumour treatment by PDT induces a marked rise in ceramide levels in cancer cells within these lesions. Cancer cells from PDT-treated SCCVII tumours undergoing apoptosis were found to have much higher ceramide levels and substantially lower S1P levels than their viable counterparts. Compared to cancer cells, considerably higher ceramide and S1P levels were consistently found in TAMs. Treatment of SCCVII tumour-bearing mice with ceramide analog LCL29 induced a rise in ceramide levels in TAMs but not in cancer cells. When combined with PDT, LCL29 treatment produced a further increase in ceramide levels in TAMs while having no evident impact on ceramide content in cancer cells within same tumours. The results highlight SLs as important participants in tumour response to PDT and potential adjuvant therapeutic targets to PDT.