Introduction: The preparation of seasonal influenza virus vaccines and especially its large-scale production requirement after the emergence or reemergence of a pandemic will need an alternative host cell system due to current suboptimal methods and the insufficiency of embryonated chicken eggs needed for producing them. In response to the vital and increasing demand for alternative means for influenza vaccine production, a cell line culture on microcarriers could be a potential alternative to the egg-based production. Methods: Influenza A/PR/8/1934 H1N1 was purified and quantified by plaque assay. The purified virus with 0.01 multiplicity of infection (MOI) was inoculated on Madin-Darby canine kidney-Siat1 (MDCK-SIAT1) cell line. Cytodex-1 microcarrier beads (2 g/l and 2.0×10⁵cells/ml) were used in a spinner flask to culture MDCK-SIAT1cells .The culture medium was harvested and clarified and the virus yield was quantified by 50% cell culture infective dose ( CCID₅₀) and hemagglutination assays. Next, the virus was concentrated and purified by ultra-filtration and ultra-centrifugation, respectively. Results: MDCK-SIAT1cells attached to the microcarriers and the cell numbers were increased efficiently. The cellular yield from the microcarrier culture was 2×10⁶ cells/ml after 4-5 days. The yield of the virus titer measured by CCID₅₀ and hemagglutination assays after the clarification was 10⁸ CCID₅₀/ml and 40960 HA unit/ml, respectively. Conclusion: MDCK-SIAT1cells may be considered as a new substrate for the production of influenza vaccines. Using Cytodex-1 microcarrier beads can be an appropriate strategy to improve the viral yield and to lower the cost of influenza vaccine production. Vac Res , 2014, 1 (1): 36-40