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Background Hundreds of microRNAs (miRNAs), comprising small non‐coding RNAs of 20–24 nucleotides, have been discovered, although the entirety of their biological functions is poorly understood. Overexpression or suppression approaches are commonly performed to investigate the function of specific miRNAs. In the present study, we focused on generating a lentiviral vector‐based strategy that enables hsa‐miR‐223‐3p (miR‐223) overexpression and suppression in the target cells for functional analysis of this miRNA easily and rapidly. Methods The sequence that gives rise to miR‐223 and the sequence generating the sponge RNA with four binding sites for miR‐223 were cloned in pLVX‐shRNA2 vector. The functionality of the vector to overexpress miR‐223 was evaluated by quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) assays, whereas the post‐transcriptional regulation exerted by miR‐223 was evaluated by luciferase reporter assays in AD‐293 cells. The anti‐miR‐223 sponge activity with one binding site for miR‐223 (pmCherry‐anti‐miR‐223) was confirmed by qRT‐PCR and the restoration of its target (IKKα) was evaluated by western blot assays in Jurkat cells. Results The pLVX‐miR‐223 vector is functional for over‐expressing miR‐223 and regulates the mRNA of MDR1/ABCB1 at the post‐transcriptional level in AD‐293 cells. The anti‐miR‐223 sponge with one miR‐223 binding site efficiently modulates the miR‐223 availability and not the one with four sites. The over‐expression of anti‐miR‐223 correlated with a decrease in the levels of miR‐223 and, consequently, with an increase in the expression level of the IKKα protein in Jurkat cells. Conclusions This single miRNA and miRNA sponge expression system specifically alters the availability of miR‐223 in mammalian cells.