Abstract: To construct eukaryotic expression vector of EGFL6 and transient transform HEK293T cell to investigate EGFL6 effects on tumor cells with EGFL6 conditioned medium. EGFL6 gene fragments and enhanced green fluorescence protein（EGFP）gene fragments were sub-cloned by PCR and combined to construct EGFP-EGFL6 fusion protein gene fragment by overlapping PCR. The sequencing identified EGFP gene fragments and EGFP-EGFL6 gene fragments were inserted to eukaryotic expression vector pAAV to construct the expression vectors. Purified by endotoxin-free kit, the expression vector plasmids were transiently transformed into HEK293T cells. The expression of EGFL6 fusion protein was identified by fluorescence localization and Westernblot analysis. The effects of EGFL6 on tumor cell proliferation of A375 and SKOV3 were assayed by CCK-8 reagent using EGFL6 expression conditioned medium. The restriction enzyme assay showed that the expression vectors of pAAV-EGFP and pAAV-EGFP-EGFL6 were constructed successfully. EGFP fluorescence localization and western blot assay results showed that EGFP-EGFL6 fusion protein was expressed successfully in HEK293T cells. The cell proliferation assay showed that EGFL6 enhanced tumor cell proliferation of A375 and SKOV3 and the cell proliferation rates reached about（37.79±14.05）% and（30.53±6.31）%. EGFL6 was expressed in HEK293T cells and the EGFL6 expression conditioned medium could promote tumor cell proliferation.