To investigate the effect of 5-aminolevulinic acid (ALA) mediated photodynamic therapy (PDT) on the invasion and metastasis in cutaneous squamous cell carcinoma (cSCC) cell line(SCL-1) and to study whether the effect was via the MTSS1 gene and p63 gene related pathways. SCL-1 cells were cultured and submitted to ALA-PDT treatment (ALA-PDT group), ALA treatment alone (ALA group), LED illumination alone (LED group) and remains untreated (control group). Scratch test, Transwell migration chamber assay and Matrigel cell invasion assay were used to detect the ability of migration and invasion of SCL-1 cells after treatment. The mRNA levels and protein expressions of tumor metastasis suppressor gene (MTSS1) and p63 gene were further detected by using quantitative real-time PCR and flow cytometry assay respectively after treatment. The migration and invasion abilities of SCL-1 cells after treatment were significantly reduced in the ALA-PDT groups than that in ALA group, LED group and control group (P<0.05). Both the mRNA and protein expression levels of MTSS1 gene were up-regulated, while the mRNA and protein expression levels of p63 gene were down-regulated after ALA-PDT treatment. ALA-PDT suppressed the migration and invasion of human cSCC cell line, probably via the MTSS1 gene and p63 gene related pathways. This study put forward a possible mechanism of invasion in SCL-1 cell, also providing a potential target for the therapy of cSCC.