The red flour beetle, Tribolium castaneum follows XX (female) and XY (male) sex determination system. Maternal supply of Transformer (Tra) is required for XX insects to follow the female pathway. The nature and the source of signal that regulates male sex determination in XY beetles are not known. Parental RNAi aided knockdown in expression of tra masculinizes genetic females (XX) that are fertile. The virgin females mated with these masculinized genetic females produced all female progeny. We present the genetic evidence to show that the factor responsible for male sex determination is present on Y chromosome. These data also suggest that Y chromosome in T. castaneum is not required for male fertility.