A matrix A is called totally positive (or totally non-negative) of order k, denoted by T P k (or T N k ), if all minors of size at most k are positive (or non-negative). These matrices have featured in diverse areas in mathematics, including algebra, analysis, combinatorics, differential equations and probability theory. The goal of this article is to provide a novel connection between total positivity and optimization/game theory. Specifically, we draw a relationship between totally positive matrices and the Linear Complementarity Problem (LCP), which generalizes and unifies linear and quadratic programming problems and bimatrix games -this connection is unexplored, to the best of our knowledge. We show that A is T P k if and only if for every submatrix Ar of A formed from r consecutive rows and r consecutive columns (with r ≤ k), LCP(Ar, q) has a unique solution for each vector q < 0. In fact this can be strengthened to check the solution set of LCP at a single vector for each such square submatrix. These novel characterizations are in the spirit of classical results characterizing T P matrices by Gantmacher-Krein [Compos. Math. 1937] and P -matrices by Ingleton [Proc. London Math. Soc. 1966].Our work contains two other contributions, both of which characterize TP using single test vectors whose co-ordinates have alternating signs -i.e., lie in a certain open bi-orthant. First, we improve on one of the main results in recent joint work [Bull. London Math. Soc., in press], which provided a novel characterization of T P k matrices using sign non-reversal phenomena. We further improve on a classical characterization of total positivity by Brown-Johnstone-MacGibbon [J. Amer. Statist. Assoc. 1981] (following Gantmacher-Krein, 1950) involving the variation diminishing property. Finally, we use a Pólya frequency function of Karlin [Trans. Amer. Math. Soc. 1964] to show that our aforementioned characterizations of total positivity, involving (single) test-vectors drawn from the 'alternating' bi-orthant, do not work if these vectors are drawn from any other open orthant.