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Objective: The purpose of the present study was to determine whether type III IFN can modulate the autophagic response in human osteosarcoma cell., Methods: Human osteosarcoma cell were treated with Interferon-λ1. We investigated that Interferon-λ1 could inhibit the invasive ability of osteosarcoma cells by Matrigel invasion assay. Autophagy were assessed by acridine orange staining, MDC staining and Transmission electron microscopy., Results: In this study, we found that Interferon-λ1 could inhibit the invasive ability of osteosarcoma cells. Acridine orange staining and MDC staining showed that Interferon-λ1 triggered the accumulation of acidic vesicular and autolysosomes in osteosarcoma cell. The acridine orange osteosarcoma cell ratios were 3.6 ± 0.5%, 4.5 ± 0.8%, and 12.4 ± 1.7% after treatment with 1, 10, and 100 ng/mL Interferon-λ1 for 48 h. Osteosarcoma cell cells treated with 100 ng/mL Interferon-λ1 for 48 h developed autophagy some-like characteristics, including single or double-membrane vacuoles containing intact and degraded cellular debris., Conclusions: Interferon-λ1 could inhibit the invasive ability of osteosarcoma cells. Autophagy can be induced in a dose-dependent manner by treatment with Interferon-λ1 in osteosarcoma cell.