microRNAs　(miRNAs)　act　as　a　major　regulator　of　acquired　chemo-resistance　in　various　types　of　cancer　therapeutics.　This　study　investigated　the　contribution　of　miRNAs　in　influencing　multiple　drug　resistance　in　esophageal　squamous　cell　carcinoma　(ESCC).　The　sensitivity　of　four　ESCC　cell　lines　(EC109,　EC9706,　TE-1　and　KYSE-150)　to　5-fluorouracil　(5-FU)　and　oxaliplatin　(OX)　was　determined　by　MTT　assay.　A　5-FU　and　OX-resistant　subline,　EC9706R,　was　established　by　continuous　exposure　to　stepwise　increasing　concentration　of　5-FU　and　OX.　Microarray　technology　was　used　to　compare　the　differential　expression　of　miRNAs　between　resistant　cells　and　parental　cells.　Chemo-sensitivity　assay　was　performed　to　evaluate　drug　response　in　EC9706R　cells　transfected　with　miRNA　mimic　or　inhibitor.　The　direct　targets　of　miRNA　were　identified　by　employing　pathway　analysis　and　then　confirmed　with　luciferase　assay.　Sixty　ESCC　tissue　samples　and　their　paired　adjacent　normal　tissues　were　collected　to　validate　the　expression　of　identified　miRNA.　Mouse　models　were　further　utilized　to　investigate　the　function　of　miRNA　on　acquired　chemo-resistance.　MicroRNA　panel　results　indicated　that　a　total　of　12　miRNAs　were　differentially　expressed　and　miR-141-3p　was　highly　over　expressed　in　resistant　cells.　Inhibition　of　miR-141-3p　reversed　acquired　chemo-resistance　in　EC9706R　cells　by　stimulating　apoptosis.　The　expression　of　miR-141-3p　was　significantly　increased　in　ESCC　tissue　samples　compared　to　their　matched　distant　normal　tissues.　In　addition,　the　elevated　miR-141-3p　expression　was　found　to　be　associated　with　ESCC　differentiation　status　and　TNM　stage.　Moreover,　Phosphatase　and　tensin　homolog　(PTEN)　was　identified　as　direct　target　of　miR-141-3p.　Western　blot　exhibited　altered　protein　levels　of　PTEN,　Akt,　and　PI3k　with　miR-141-3p　inhibitor.　An　inverse　correlation　between　PTEN　expression　and　miR-141-3p　expression　was　also　observed　in　tissue　samples.　EC9706R　xenograft　mouse　model　became　sensitized　to　5-FU　and　OX　treatment　following　miR-141-3p　inhibitor　transfection　in　vivo.　Our　study　demonstrated　that　miR-141-3p　contributed　to　an　acquired　chemo-resistance　through　PTEN　modulation　both　in　vitro　and　in　vivo.