Background: Leptospirosis is one of the most important contagious zoonosis diseases caused by different serovars of Leptospira interrogans. Objectives: The aim of the present study is to determine the prevalence rate of Leptospira pathogen serovars in cows and relation to human population in Gilan province during 2003-2013. Methods: Totally 1250 serum samples were collected from different cities in Gilan (800 samples at 2003 and 450 samples at 2013 from cows). The serum samples were analyzed employing the Microscopic Agglutination Test (MAT) at the Razi Research Institute Center. Results: The infection rate was high, in 3-4 years old cows and in spring seasons. In 2013, serum samples showed a positive reaction against leptospira which was higher compared to 2003. The most seroprevalent serovar was Grippotyphosa and the least was serovar Canicola in 2003 and predominant seroprevalent was Icterohaemprragia and the least was Pomona in 2013. The most percentage rates of MAT titers were 1:200 in 2003, and most percentage rates of MAT titers were 1:800 and1:1600 in 2013. The infection rate was high and according to the review of Leptospirosis prevalence in cows and its relation to the human population between 2003 and 2013. The relationship between human infection and an increase in animal titer with the highest rate of infection seen in the countryside. Conclusions: There is an important concern about the possibility of the occurrence of an acute form of the disease with low prevalence. There is, therefore, a need to control the disease in Gilan Province.