Abstract: Endophytic bacteria of Spinacia oleracea with spraying and without spraying acephate were isolated with classical techniques. The effects of pesticide on the endophytic bacteria were analyzed based on 16S rRNA sequences. The results showed that spraying the acephate had significantly decreased the number of endophytic bacteria in S.oleracea leaves, whereas the number of endophytic bacteria in S.oleracea roots had no significantly difference between spraying acephate and no spraying.24 endophytic bacterias were isolated from S.oleracea with spraying acephate and identified as 6 genera and 7 species, 3 other isolates were possible new species or genus, 2 characteristics strains were Pseudomonas monteilii and Arthrobacter arilaitensis. 35 endophytic bacterias were isolated from S.oleracea without spraying acephate and identified as 14 genera and 16 species, 6 other isolates were possible new species or genus. The diversity of culturable endophytic bacteria was reduced with spraying acephate.