To replicate, HIV-1 needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a sub-unit of the SWI/SNF chromatin remodeling complex, was the first co-factor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-LTR circle and integrated DNA forms accumulate upon SNF5/Ini1 knock-down. By yeast two-hybrid, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication, and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.