Gallbladder carcinoma (GBC) is the most common malignant tumour in the biliary tract, but effective therapeutics are lacking. Based on our previous studies, miR-135a is a potential tool to inhibit GBC proliferation. In this study, we constructed miR-135a-loaded DSPE-PEG2000 liposomes modified with Anti-EGFR antibodies (Anti-EGFR-CIL-miR-135a). The results of an analysis of their physicochemical properties indicated the particle size of it was 222.0 ± 2.1 nm in diameter with an uptake efficiency of 86.5%. Next, the post-treatment biological behaviours of GBC, specifically, invasion, metastasis and apoptosis, were evaluated. miR-135a inhibited GBC invasion and metastasis and promoted apoptosis compared to controls. Additionally, miR-135a targeted and regulated the expression of ROCK1, HOXA10 and BCL-2. Due to the targeted effects of Anti-EGFR-CIL-miR-135a, the GBC tumour growth rate was 60% lower in an in vivo xenograft-bearing mouse model compared to controls. Thus, Anti-EGFR-CIL-miR-135a is a promising therapeutic strategy to combat GBC.